Methods for screening cellular proliferation using isotope labels

ABSTRACT

The present invention relates to methods for measuring the proliferation and destruction rates of cells by measuring deoxyribonucleic acid (DNA) synthesis and/or destruction. In particular, the methods utilize non-radioactive stable isotope labels to endogenously label DNA synthesized through the de novo nucleotide synthesis pathway in a cell. The amount of label incorporated in the DNA is measured as an indication of cellular proliferation. The decay of labeled DNA over time is measured as an indication of cellular destruction. Such methods do not involve radioactivity or potentially toxic metabolites, and are suitable for use both in vitro and in vivo. Therefore, the invention is useful for measuring cellular proliferation or cellular destruction rates in humans for the diagnosis, prevention, or management of a variety of disease conditions in which cellular proliferation or cellular destruction is involved. The invention also provides methods for measuring proliferation or destruction of T cells in a subject infected with human immunodeficiency virus (HIV) and methods of screening an agent for a capacity to induce or inhibit cellular proliferation or destruction. In addition, the invention provides methods for measuring cellular proliferation in a proliferating population which utilize both radioactive isotope labels and stable isotopes to endogenously label DNA through the de novo nucleotide synthesis pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Pat. Ser. No.09/440,596 filed Nov. 15, 1999, now U.S. Pat. No. 6,461,806 which was aDivisional Application of U.S. application Ser. No. 09/075,309 filed May8, 1998 (now U.S. Pat. No. 6,010,846), which was a Continuation-in-PartApplication of U.S. Ser. No. 08/857,007 filed May 15, 1997, (now U.S.Pat. No. 5,910,403), which is incorporated herein by reference in itsentirety for all purposes. This invention was supported in part by agrant from the National Institutes of Health (Grant No. AI-41401). TheGovernment may have certain rights in this invention.

This invention was supported in part by a grant from the NationalInstitutes of Health (Grant No. AI-41401). The Government may havecertain rights in this invention.

1. INTRODUCTION

The present invention relates to methods for measuring the proliferationand destruction rates of cells by measuring deoxyribonucleic acid (DNA)synthesis. In particular, the methods utilize non-radioactive stableisotope labels to endogenously label DNA synthesized through the de novonucleotide synthesis pathway in a cell. The amount of label incorporatedin the DNA is measured as an indication of cellular proliferation. Suchmethods do not require radioactivity or potentially toxic metabolites,and are suitable for use both in vitro and in vivo. Therefore, theinvention is useful for measuring cellular proliferation and/or cellulardestruction rates in humans for the diagnosis of a variety of diseasesor conditions in which cellular proliferation or destruction isinvolved. The invention also provides methods of screening an agent fora capacity to induce or inhibit cellular proliferation or cellulardestruction and methods for measuring the proliferation or destructionof T cells in a subject infected with human immunodeficiency virus(HIV).

2. BACKGROUND OF THE INVENTION

Control of cell proliferation is important in all multicellularorganisms. A number of pathologic processes, including cancer andacquired immunodeficiency syndrome (AIDS) (Ho et al., 1995, Nature373:123-126; Wei et al., 1995, Nature 373:117-122; Adami et al., 1995,Mutat. Res. 333:29-35), are characterized by failure of the normalregulation of cell turnover. Measurement of the in vivo turnover ofcells would therefore have wide applications, if a suitable method wereavailable. Prior to the present invention,direct and indirect techniquesfor measuring cell proliferation or destruction existed, but both typeswere flawed.

Direct measurement of cell proliferation generally involves theincorporation of a labeled nucleoside into genomic DNA. Examples includethe tritiated thymidine (³H-dT) and bromodeoxyuridine (BrdU) methods(Waldman et al., 1991, Modern Pathol. 4:718-722; Gratzner, 1982, Science218:474475). These techniques are of limited applicability in humans,however, because of radiation induced DNA damage with the former (Asheret al., 1995, Leukemia and Lymphoma 19:107-119) and toxicities ofnucleoside analogues (Rocha et al., 1990, Eur. J. Immunol. 20:1697-1708)with the latter.

Indirect methods have also been used in specific cases. Recent interestin CD4⁺ T lymphocyte turnover in AIDS, for example, has been stimulatedby indirect estimates of T cell proliferation based on their rate ofaccumulation in the circulation following initiation of effectiveanti-retroviral therapy (Ho et al., 1995, Nature 373:123-126; Wei etal., 1995, Nature 373:117-122). Unfortunately, such indirect techniques,which rely on changes in pool size, are not definitive. The increase inthe blood T cell pool size may reflect redistribution from other poolsto blood rather than true proliferation (Sprent and Tough, 1995, Nature375:194; Mosier, 1995, Nature 375:193-194). In the absence of directmeasurements of cell proliferation, it is not possible to distinguishbetween these and other (Wolthers et al., 1996, Science 274:1543-1547)alternatives.

Measurement of cell proliferation is of great diagnostic value indiseases such as cancer. The objective of anti-cancer therapies is toreduce tumor cell growth, which can be determined by whether tumor DNAis being synthesized or being broken down. Currently, the efficacy oftherapy, whether chemotherapy, immunologic therapy or radiation therapy,is evaluated by indirect and imprecise methods such as apparent size byx-ray of the tumor. Efficacy of therapy and rational selection ofcombinations of therapies could be most directly determined on the basisof an individual tumor's biosynthetic and catabolic responsiveness tovarious interventions. The model used for bacterial infections inclinical medicine—culture the organism and determine its sensitivitiesto antibiotics, then select an antibiotic to which it is sensitive—couldthen be used for cancer therapy as well. However, current managementpractices proceed without the ability to determine directly how well thetherapeutic agents are working.

A long-standing vision of oncologists is to be able to selectchemotherapeutic agents the way antibiotics are chosen—on the basis ofmeasured sensitivity to each drug by the tumor of the patient inquestion. The ability to measure cancer cell replication would placechemotherapy selection and research on an equal basis as antibioticselection, with great potential for improved outcomes.

Accordingly, there remains a need for a generally applicable method formeasuring cell proliferation that is without hazard and can be appliedin the clinical arena.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for measuring cellularproliferation and/or destruction rates by measuring DNA synthesis. Inparticular, it relates to the use of a non-radioactive stable isotopelabel to endogenously label DNA synthesized by the de novo nucleotidesynthesis pathway in a cell. The label incorporated into the DNA duringDNA synthesis is readily detectable by methods well known in the art.The amount of the incorporated label can be measured and calculated asan indication of cellular proliferation and destruction rates.

The invention is based, in part, on the Applicants' discovery that DNAsynthesis can be measured by labeling the deoxyribose ring with a stableisotope label through the de novo nucleotide synthesis pathway. Cellularproliferation was measured in vitro, in an animal model and in humans.In vitro, the proliferation of two cell lines in log phase growth wasmeasured by the methods of the invention and was shown to be in closequantitative agreement with the increased number of cells by direct cellcounting, which is considered the least ambiguous measure of cellproliferation. In animals, the methods of the invention were also shownto be consistent with values estimated previously by independenttechniques. For example, thymus and intestinal epithelium were shown tobe rapid turnover tissues, while turnover of liver cells was muchslower. In humans, the observed pattern of a lag phase followed by rapidappearance of a cohort of labeled granulocytes is also consistent withprevious observation.

The methods differ from conventional labeling techniques in 3 majorrespects. First, conventional isotopic methods label DNA through theknown nucleoside salvage pathway, whereas the methods of the inventionlabel deoxyribonucleotides in DNA by the known de novo nucleotidesynthesis pathway (FIG. 1), through which purine and pyrimidinenucleotides are formed. In brief, in the de novo nucleotide synthesispathway, ribonucleotides are formed first from small precursor molecules(e.g., glucose, glucose-6-phosphate, ribose-5-phosphate, purine andpyrimidine bases, etc.) and are subsequently reduced todeoxyribonucleotides by ribonucleotide reductase. See, e.g., FIG. 1;Reichard, 1988, Ann. Rev. Biochem. 57:349-374 (e.g., FIGS. 1 and 2);THOMAS SCOTT & MARY EAGLESON, CONCISE ENCYCLOPEDIA BIOCHEMISTRY 406-409,501-507 (2d ed. 1988); and TEXTBOOK OF BIOCHEMISTRY WITH CLINICALCORRELATIONS (Thomas M. Devlin ed., 3d ed. 1992)), each of which isincorporated herein in its entirety for all purposes. Through the actionof ribonucleotide reductase, three deoxyribonucleotides, DADP, dCDP, anddGDP, are produced directly. These deoxyribonucleotides are thenphosphorylated by nucleoside diphosphate kinase to form correspondingdeoxyribonucleotide triphosphates—DATP, dCTP, and dGTP. A fourthdeoxyribonucleotide, dTTP, is also formed from ribonucleotide reductase,after additional remodeling. The four deoxyribonucleotidetriphosphates—dATP, dCTP, dGTP, and dTTP—are utilized to synthesize DNA.FIG. 1, which illustrates the de novo nucleotide synthesis pathway, alsoshows the pathway for endogenous labeling of DNA from stableisotope-labeled glucose.

Labeling via the de novo nucleotide synthesis pathway is advantageousbecause in most cells that enter the S-phase of the cell cycle, the keyenzymes controlling de novo synthesis ofdeoxyribonucleotide-triphosphates (dNTP's), in particular ribonucleotidereductase (RR), are upregulated, whereas the enzymes of the nucleosidesalvage pathway (which represents an alternative pathway for formationof purine and pyrimidine nucleotides) are suppressed (Reichard, 1978,Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374; Cohenet al., 1983, J. Biol. Chem. 258:12334-12340; THOMAS SCOTT & MARYEAGLESON, CONCISE ENCYCLOPEDIA BIOCHEMISTRY 543-544 (2d ed. 1988).

Second, the label can be detected in the methods of the invention inpurine deoxyribonucleosides instead of pyrimidines (e.g., from ³H-dT orBrdU). This is advantageous because the de novo synthesis pathway tendsto be more active for purine than pyrimidine dNTP's (Reichard, 1978,Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374; Cohenet al., 1983, J. Biol. Chem. 258:12334-12340). In fact, regulatorydeoxyribonucleotides have been shown in lymphocytes (Reichard, 1978,Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374) toexert negative feedback on RR for pyrimidine dNTP synthesis but positivefeedback for purine dNTP synthesis, ensuring that the de novo synthesispathway is always active for the purines but is variable for thepyrimidines.

Additionally, the methods of the invention which use stable isotopelabels instead of non-stable radio-isotopes are safe for human use.Therefore, a wide variety of uses are encompassed by the invention,including, but not limited to, measurement of the rate of cellularproliferation and/or destruction in conditions where such information isof diagnostic value, such as cancer, AIDS, hematologic disorders,endocrine disorders, bone disorders and organ failure. Where non-toxicstable isotopes are employed, such cellular proliferation anddestruction rates can be measured in vivo in a subject.

In one aspect, the invention provides methods for measuring cellularproliferation or cellular destruction rates which comprise contacting acell with a detectable amount of a stable isotope label which isincorporated into DNA via the de novo nucleotide synthesis pathway, anddetecting the label in the DNA.

The invention also provides methods for measuring the rates of cellularproliferation and/or cellular destruction in a subject. Such methodscomprise contacting a cell with a detectable amount of a stable isotopelabel which is incorporated into DNA via the de novo nucleotidesynthesis pathway and detecting the label in the DNA of the subject.

In another aspect of the invention, methods for measuring the rates ofproliferation and/or destruction of T cells in a subject infected withhuman immunodeficiency virus (HIV) are provided. Such methods compriseadministering a detectable amount of a stable isotope label to thesubject, wherein the label is incorporated into DNA of the T cells ofthe subject via the de novo nucleotide synthesis pathway. The label inthe DNA of the T cells of the subject is detected to measure the ratesof proliferation and/or destruction of T cells in the subject.

The invention also provides methods of screening an agent for a capacityto induce or inhibit cellular proliferation. Such methods comprisecontacting a cell with the agent, contacting the cell with a detectableamount of a stable isotope label which is incorporated into DNA of thecell via the de novo nucleotide synthesis pathway, and detecting thelabel in the DNA. The amount of label, compared to a control applicationin which the cell is not exposed to the agent, indicates the extent ofcellular proliferation and thereby whether the agent induces or inhibitscellular proliferation.

In another aspect, the invention provides methods of screening an agentfor a capacity to induce or inhibit cellular proliferation in a subjectexposed to the agent. Such methods comprise exposing the subject to theagent; administering a detectable amount of a stable isotope label tothe subject, wherein the label is incorporated into DNA of the subjectvia de novo nucleotide synthesis pathway; and detecting the label in theDNA of a cell of interest in the subject indicating cellularproliferation in the subject. The amount of label relative to a controlapplication in which the subject is not exposed to the agent indicatesthe extent of cellular proliferation and thereby whether the agentinduces or inhibits cellular proliferation in the subject.

In yet another aspect of the invention, methods for measuring cellularproliferation in a proliferating or dividing population of cells areprovided. These methods comprise: (a) contacting the proliferatingpopulation of cells with a detectable amount of a first label, whereinthe first label comprises a stable isotope label which is incorporatedinto DNA via the de novo nucleotide synthesis pathway; (b) detecting thefirst label incorporated into the DNA to measure cellular proliferationin the proliferating population of cells; (c) contacting theproliferating population of cells with a detectable amount of a secondlabel, wherein the second label comprises a radioactive isotope labelwhich is incorporated into DNA via the de novo nucleotide synthesispathway; and (d) detecting the second label incorporated the DNA tomeasure cellular proliferation in the proliferating population of cells.In some such methods employing both radioactive and non-radioactiveisotope labels, steps (a) and (b) are performed before steps (c) and(d). In other such methods utilizing both radioactive andnon-radioactive isotope labels, steps (c) and (d) are performed beforesteps (a) and (b). Alternatively, in some such methods, steps (a) and(c) can be performed simultaneously and steps (b) and (d) can beperformed simultaneously.

The present invention also includes methods for determining thesusceptibility of a subject to a disease or disorder (includingdisorders which are not yet themselves disease, but which predispose thesubject to a disease) which induces a change in a rate of cellularproliferation in the subject. Such methods comprise exposing the subjectto a condition or an agent which can produce or induce the disease ordisorder and administering a detectable amount of a stable isotope labelto the subject. The label is incorporated into DNA of the subject via denovo nucleotide synthesis pathway. The label in the DNA of the subjectis detected. An increase in label in the DNA of the subject, compared toa control application in which the subject is not exposed to thecondition or agent, indicates an increase in the rate of cellularproliferation and susceptibility of the subject to the disease ordisorder.

The invention also includes methods for determining the susceptibilityof a subject to a disease which induces a change in a rate of cellulardestruction (including disorders which are not yet themselves disease,but which predispose the subject to a disease) in a subject. Thesemethods comprise exposing the subject to a condition or an agent whichcan produce the disease, administering a detectable amount of a stableisotope label to the subject, which label is incorporated into DNA ofthe subject via de novo nucleotide synthesis pathway, and detecting thelabel in the DNA of the subject. A loss of label in the DNA of thesubject compared to a control application in which the subject is notexposed to the condition or agent indicates an increase in the rate ofcellular destruction and susceptibility of the subject to the disease.

In another aspect, the invention provides methods of labeling DNA in acell which comprise contacting the cell with a detectable amount of astable isotope label which is incorporated into DNA via de novonucleotide synthesis pathway.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Biochemistry of DNA synthesis and routes of label entry. Not allintermediates are shown. G6P, glucose-6-phosphate; R5P,ribose-5-phosphate; PRPP, phosphoribosepyrophosphate, DNNS, de novonucleotide synthesis pathway; NDP, ribonucleoside-diphosphates: RR,ribonucleoside reductase; dN, deoxyribonucleoside; dNTP,deoxyribonucleoside-triphosphate; ³H-dT, tritiated thymidine; BrdU,bromodeoxyuridine.

FIG. 2: Overview of a specifically exemplified method for measurement ofDNA synthesis by incorporation of [6,6-²H₂] glucose.

FIG. 3: GC-MS of DNA digest (total ion current). Mass spectra of purinedeoxyribonucleoside dA and dG peaks are shown as insets.

FIGS. 4A-4D: Labeling of tissue culture cells during log-phase growth invitro. Enrichment of dA from cellular DNA in hepatocyte (HepG2) (4A) andlymphocyte (H9) (4B) cell lines grown in media enriched with [6,6-²H₂]glucose. Lymphocyte data include results from experiments at twodifferent glucose enrichments. FIGS. 4C and 4D are comparisons offractional synthesis of DNA by HepG2 cells (4C) and H9 cells (4D),calculated from M₂ enrichments of dA/medium glucose, with increase incell numbers by counting.

FIGS. 5A-5B: Enrichment of dA from DNA of (FIG. 5A) hepatocyte (HepG2)and (FIG. 5B) lymphocyte (H9) cell lines grown in media containing 100%[6,6-²H₂] glucose for prolonged periods with repeated subcultures.

FIGS. 6A-6B: FIG. 6A demonstrates the enrichment of M₅ ion ofdeoxyadenosine from DNA of hepatocyte cell line (HepG2) cells grown inapproximately 20% [U-¹³C₆] glucose. FIG. 6B is a comparison offractional synthesis of DNA from M₅ labeling to proportion of new cellsby direct counting.

FIG. 7: Fractional synthesis of granulocytes in peripheral blood from 4subjects following two-day infusion of [6,6-²H₂] glucose, commencing attime zero. Open symbols, control subject; closed symbols, HIV-infectedsubjects. Fraction of new cells was calculated by comparison of dAenrichment to average plasma glucose enrichment, after correcting forestimated 35% intracellular dilution.

FIG. 8: Fractional synthesis of mixed lymphocytes (including B and Tcells) obtained from peripheral blood of an HIV-infected patientfollowing two-day infusion of [6,6-²H₂] glucose.

FIGS. 9A-9C: FIG. 9A shows a fluorescence-activated cell sorting (FACS)isolation of purified peripheral blood CD4⁺ and CD8⁺ T lymphocytes. Forseparation by FACS, 50-70 milliliters (mls) of peripheral blood wasfractionated by ficoll-hypaque gradient sedimentation to obtain about50-100×10⁶ peripheral blood mononuclear cells (PBMCs). These cells werestained within 4 hours with phycoerythrin-Cychrome 5 (PE-Cy5)-conjugatedanti-CD4 and allophycocyanin (APC)-conjugated anti-CD8 antibodies andsubjected to sort purification on a dual laser (argon 310 nm, argon 488nm) FACS Vantage (Becton Dickinson Immunocytometry Systems, San Jose,Calif.) equipped for biocontained procedures with viable, HIV-1-infectedcells (upper panel). For kinetic analysis by GC-MS, it was optimal toobtain at least one million cells each of the purified CD4⁺ and CD8⁺ Tcell subpopulations. Resort analysis showed sort purities of >98%(middle panel and lower panel for CD4⁺ and CD8⁺ T cells, respectively).FIG. 9B shows GC-MS of derivatized dA prepared from T cell DNA. DNA wasextracted from cells with a QUIamp blood kit (Quiagen, Valencia,Calif.). The DNA was subjected to enzymatic hydrolysis using nuclease P1followed by snake venom phosphodiesterase I and alkaline phosphatase(Macallan et al., 1998, Proc. Natl. Acad. Sci. USA 95:708). Digested DNAwas separated by HPLC (reverse phase C-18 Vydac column; buffer A, 2.5%methanol; buffer B, 50% methanol; 1 ml/min (milliliters/minute) flowrate with gradient 0% B to 8% B over 10 min, then 8% B to 100% B over 10min and maintenance at 10% B for final 10 min; OD (optical density) 260nm monitored), and the dA peak collected (at about 20 min). Afterevaporation of methanol under N₂, the dA was derivatized by acetylationwith acetonitrile:acetic anhydride:N-methylimidazole (100:10:1) for 60minutes at room temperature, evaporation to dryness, and methylationwith CH₃Cl. For GC-MS of dA, an HP model 5971 MS with 5890 GC andautosampler (Hewlett-Packard, Palo Alto, Calif.) was used with a DB-5MSor Restek Rtx-5 amine column. Injector temperature was 320° C., initialoven temperature 140° C. for 2 min then rising to 300° C. at 40°/min andmaintained at 300° C. for 10 min. Electron impact ionization andselected ion monitoring mode were used. The ions monitored were m/z 276and 278, representing the molecular ion of acetylated dA minus oneacetate. FIG. 9C shows standard curve of ²H-dA. Sample enrichments werecalculated by comparison to abundance—corrected standard curves using[²H₂]dA. Weighed mixtures of standard [²H₂]dA (Isotec, Miamisburg, Ohio)and natural abundance dA were injected at different volumes to span theabundances of dA potentially present in samples. A standard curve wasthen matched to each sample's measured abundance.

FIG. 10: The time course of CD4⁺ T cell labeling after 48 hr iv infusionof ²H-glucose (results shown from group III—subjects in whom the mostrepeat measurements were performed). The highest value observed was usedto calculate fractional replacement (new cells present). [6,6-²H₂]Glucose (60-100 grams (g), Isotec Inc, Miamisburg, Ohio) wasadministered iv in one liter 0.45% saline, infused over 48 hr at a rateof 1.25 to 2.0 grams/hour (g/hr). Subjects were maintained on eucaloric,carbohydrate restricted diets (<50 g carbohydrate per day) for the 48 hrinfusion period, to allow maximal plasma glucose enrichments.

FIGS. 11A-11B: FIG. 11A shows a absolute proliferation rates(cells/μL/day) of blood CD4⁺ and CD8⁺ T cells in different groups.Numbers represent number of subjects per group. FIG. 11B shows values ofk(d⁻¹) (rate constant) for blood CD4⁺ and CD8⁺ T cells in differentgroups. Numbers in represent number of subjects per group. Symbols: a,p<0.05 vs. normals; b, p<0.05 vs. HIV+ (HIV positive); c, p<0.05 vs.short-term highly active anti-retroviral therapy (HAART); d, p<0.05 vs.short-term HAART, viral responders. For comparison of CD4⁺ vs. CD8⁺ Tcells, the only significant differences were for absolute proliferationrates in short-term HAART and short-term HAART, viral responders(p<0.05).

FIG. 12: Comparison of net accumulation rate of CD4⁺ T cells over thefirst 6 weeks after initiation of HAART regimen (addition ofritonavirlsaquinavir) to the steady-state absolute replacement rate ofCD4⁺ T cells at week 12 of HAART. Net accumulation rate was calculatedfrom the difference between baseline CD4 count (average of 2-3 values)and week 6 CD4 count. This represents the average accumulation rate oversix weeks, still lower than the measured steady-state replacement rate(19.2±15.4 cells/μL/day). A different symbol (e.g., closed square, opendiamond, open triangle, open circle, “x” symbol) designates anindividual subject. Values obtained by the two methods for eachindividual subject are connected by a line. The two solid horizontalbars represent average values for each of the two methods, respectively.

FIGS. 13A-13G: FIG. 13A shows correlation between absolute proliferationrates of blood CD4⁺ and CD8⁺ T cells in HIV+, short-term HAART andlong-term HAART subjects (r²=0.69, p<0.001). HIV+ subjects (Group II),closed diamond symbols; Short-term HAART subjects, (Group III), opensquare symbols; Long-term HAART subjects (Group IV), closed squaresymbols. FIG. 13B shows correlation between k(d⁻¹) for blood CD4⁺ andCD8⁺ T cells in HIV+, short-term HAART and long-term HAART subjects(r²=0.66, p<0.001). Symbols as the same as in FIG. 13A. FIG. 13Cillustrates correlation between absolute proliferation rate and count ofblood CD4⁺ T cells in HIV+ subjects (Group II, r²=0.96, p<0.001). FIG.13D presents correlation between absolute proliferation rate and countof blood CD4⁺ T cells in short-term HAART subjects (Group III, r²=0.55,p<0.01). FIG. 13E depicts correlation between k (rate constant (time⁻¹))and count of blood CD4⁺ T cells in HIV+ (closed square) and short-termHAART group (closed diamond); no significant correlation is present.FIG. 13F shows the relation between plasma viral load and absoluteproliferation rate of blood CD4⁺ T cells in HIV+, short-term HAART andlong-term HAART subjects (Groups II, III and IV, Table 4). Subjects weredivided into three subgroups: viral load <500, between 500 to 30,000,and >30,000 copies/ml. No differences between subgroups are present.FIG. 13G shows the relation between plasma viral load and k (rateconstant (time⁻¹)) of CD4⁺ T cells in Groups II, III and IV. Subjectswere divided into three subgroups, as in FIG. 13F above. No differencesbetween subgroups are present.

FIG. 14: Kinetic predictions of “high turnover” (accelerateddestruction) and “low-turnover” (regenerative failure) models of CD4⁺ Tcell depletion in HIV-1 infection. The predicted relationship betweenreplacement rate (turnover) and count of CD4⁺ T cells as well as theeffects of HAART are shown schematically (see text for discussion).

5. DETAILED DESCRIPTION OF THE INVENTION

The biochemical correlate of new cell production is DNA synthesis. DNAsynthesis is also relatively specific for cell division because“unscheduled” DNA synthesis is quantitatively minor (Sawada et al.,1995, Mutat. Res. 344:109-116). Therefore, measurement of new DNAsynthesis is essentially synonymous with measurement of cellproliferation.

In one aspect of the invention, methods for measuring the rates ofcellular proliferation and/or cellular destruction are provided. Suchmethods comprise contacting a cell with a detectable amount of a stableisotope label which is incorporated into DNA via the de novo nucleotidesynthesis pathway and detecting the label incorporated into the DNA. Asis understood by those of ordinary skill in the art, a stable isotopelabel is a non-radioactive isotope label. A radioactive isotope label isa label comprising a radio-isotope. A radio-isotope is an isotopic formof an element (either natural or artificial) that exhibitsradioactivity—the property of some nuclei of spontaneously emittinggamma rays or subatomic particles (e.g., alpha and beta rays).

With some such methods, the cellular proliferation rate of aproliferating population of cells can be measured. A proliferatingpopulation of cells is a group of cells that are dividing and producingprogeny. The amount of label incorporated in the DNA is measured as anindication of cellular proliferation. Measurement of the decay oflabeled DNA over time (i.e., measurement of the decline in signal of thelabel incorporated into the DNA) serves as an indication of cellulardestruction.

The methods for measuring DNA synthesis and/or destruction and thus cellproliferation and/or destruction rates described herein have severaladvantages over previously available methods. ³H-Thymidine is a potentanti-metabolite that has been used to kill dividing cells (Asher et al.,1995, Leukemia and Lymphoma 19:107-119); the toxicity of introducingradio-isotopes into DNA is avoided by the methods of the invention whichutilize non-radioactive stable isotopes. Thus, methods of the inventionemploying non-radioactive stable isotopes are safe and especially usefulfor measuring cellular proliferation and/or cellular destruction ratesin humans for the diagnosis, prevention, or management of diseases ordisorders which induce or inhibit cellular proliferation or cellulardestruction.

The toxicities of nucleoside analogues, e.g., BrdU, are also avoided byusing methods of the invention which permit labeling with a non-toxicphysiologic substrate (e.g., stable label) through endogenous syntheticpathways.

Isotopic contamination by non-S-phase DNA synthesis is also minimized bylabeling through the de novo nucleotide synthesis pathway, which isprimarily active during S-phase. The variability of labeled pyrimidinenucleoside salvage uptake is resolved by labeling purine dNTP's via thede novo nucleotide synthesis pathway, the pyrimidine nucleotide salvagepathway being the route by which previously used labels, such as³H-deoxythymidine or BrdU, must traverse to enter DNA (FIG. 1), turningwhat was previously a disadvantage (low purine dNTP labeling from thenucleoside salvage pathway) into an advantage (high and constant purinedNTP labeling from the de novo pathway). This is demonstrated by theconstancy of [6,6-²H₂] glucose incorporation into DNA even in thepresence of supraphysiologic extracellular concentrations ofdeoxyribonucleosides (Table 1). Possible input from free purine orpyrimidine base salvage does not dilute the ribose moiety of NTP's,because the salvage pathway for free bases, like de novo synthesis ofbases, involves combination with PRPP, which is synthesized from glucose(FIG. 1).

Moreover, re-utilization of label from catabolized DNA is avoided byanalyzing purine deoxyribonucleotides, because the deoxyribonucleosidesalvage pathway is low for purines. Die-away curves of labeled dA or dGin DNA after cessation of labeling will therefore be relativelyuncontaminated by isotope reutilization, and cell turnover should bemeasurable from decay curves as well as incorporation curves(Hellerstein and Neese, 1992, Am. J. Physiol. 263:E988-E1001).

Additionally, the methods of the invention provide a precisequantitative measure for enumerating numbers of new cells as opposed toconventional methods which only detect the relative increase or decreaseof cell numbers as compared to controls.

The present invention also provides in vivo methods for measuring theproliferation or depletion of T cells in subjects infected with HIV.These methods are of benefit in ascertaining the rate of proliferationor destruction of T cells, including CD4⁺ and CD8⁺ cells, in varioussubjects, including humans infected with the HIV virus and/or sufferingfrom AIDS. Such methods comprise endogenous labeling methods formeasuring DNA synthesis using non-radioactive (stable isotopes) withmass spectrometric techniques as described in detail herein and in theExamples below. In particular, such methods comprise administering adetectable amount of a stable non-radioactive isotope label to thesubject, wherein the label is incorporated into DNA of the subject viathe de novo nucleotide synthesis pathway. The label in the DNA isdetected to measure the proliferation or destruction of T cells. Suchmethods can be performed prior to or after anti-retroviral treatment ofthe subject for HIV infection. In this way, the effects of suchtreatments on T cell proliferation or destruction rates can be analyzed.

The invention also provides in vitro and in vivo methods for screeningan agent or compound for a capacity or ability to induce or inhibitcellular proliferation. Such screening methods are useful in identifyingparticular agents or compounds which stimulate or inhibit cellularproliferation. These methods are also useful in identifying“proliferogens”—agents or compounds which stimulate or encouragecellular proliferation or new cell production. In addition, suchscreening methods are of assistance in ascertaining whether a particularagent, substance, or compound is a potential carcinogen, because theability of an agent, substance, or compound to induce cell proliferationis an important criterion indicating that it may be a carcinogen,separate from or independently from its capacity to damage DNA (e.g.,Ames test for carcinogenicity of a substance or compound; Ames et al.,1973, Proc. Natl. Acad. Sci. USA 70:2281). Thus, these screening methodsserve as a convenient indicator of the carcinogenicity of an agent orcompound.

Screening methods of the invention are advantageous over previouslyavailable methods for a variety of reasons, including those outlined indetail above with regard to other methods of the invention. Inparticular, such screening methods are of benefit because they do notrequire the use of toxic non-stable radio-isotopes; on the contrary,non-toxic stable isotopes can be employed with the screening methods ofthe invention.

In addition, the invention provides methods of labeling DNA in a cell.Such methods comprise contacting a cell with a detectable amount of astable isotope label which is incorporated into DNA via de novonucleotide synthesis pathway. Such methods for labeling DNA are usefulfor the reasons described herein for other methods of the invention; inparticular, such methods are useful for in vivo applications which callfor detecting DNA in subjects, including humans, because they do notrequire the use of toxic non-stable radio-isotope labels. Such DNAlabeling methods are performed using the techniques and procedures asdescribed supra and infra in this disclosure regarding methods formeasuring cellular proliferation and cellular destruction rates in vitroand in vivo (e.g., contacting a cell with a detectable amount of astable isotope label, incorporating said label into DNA via the de novosynthesis pathway, and detecting the label in the DNA of the cell). Withsuch methods, the manner of administration, type of stable isotope label(e.g., [6,6-²H₂] glucose), and techniques for detection of such labels(e.g., mass spectrometry) are analogous to those described for othermethods of the invention relating to measuring cellular proliferationand destruction rates.

Although the specific procedures and methods described herein areexemplified using labeled glucose as the precursor and detection of thelabel by analyzing purine deoxyribonucleosides, they are illustrativefor the practice of the invention. Analogous procedures and techniques,as well as functionally equivalent labels, as will be apparent to thoseof skill in the art based on the detailed disclosure provided herein,are also encompassed by the invention.

5.1. Stable Isotope Labels for Use in Labeling DNA During de novoBiosynthesis of Nucleotides

The present invention relates to methods of measuring cellularproliferation by contacting a cell with a stable isotope label for itsincorporation into DNA via the de novo nucleotide synthesis pathway.Detection of the incorporated label is used as a measure of DNAsynthesis. Labeling DNA through the de novo nucleotide synthesis(endogenous) pathway has several advantages over conventional labelingmethods through the nucleoside salvage (exogenous) pathway. Theseinclude non-toxicity, specificity for S-phase of the cell cycle andabsence of re-incorporation of the label from catabolized DNA. The useof a non-radioactive label further avoids the risks of mutation.

In a specific embodiment illustrated by way of example in Section 6,infra, [6,6-²H₂] glucose, [U-¹³C₆] glucose and [2-¹³C₁] glycerol wereused to label the deoxyribose ring of DNA. Labeling of the deoxyriboseis superior to labeling of the information-carrying nitrogen bases inDNA because it avoids variable dilution sources. The stable isotopelabels are readily detectable by mass spectrometric techniques.

In a preferred embodiment of the invention, a stable isotope label isused to label the deoxyribose ring of DNA from glucose, precursors ofglucose-6-phosphate or precursors of ribose-5-phosphate. In embodimentswhere glucose is used as the starting material, suitable labels include,but are not limited to, deuterium-labeled glucose such as [6,6-²H₂]glucose, [1-²H₁] glucose, [3-²H₁] glucose, [²H₇] glucose, and the like;¹³C-1 labeled glucose such as [1-¹³C₁] glucose, [U-¹³C₆] glucose and thelike; and ¹⁸O-labeled glucose such as [1-¹⁸O₂] glucose and the like.

In embodiments where a glucose-6-phosphate precursor or aribose-5-phosphate precursor is desired, a gluconeogenic precursor or ametabolite capable of being converted to glucose-6-phosphate orribose-5-phosphate can be used. Gluconeogenic precursors include, butare not limited to, ¹³C-labeled glycerol such as [2-¹³C₁] glycerol andthe like, a ¹³C-labeled amino acid, deuterated water ²H₂O) and¹³C-labeled lactate, alanine, pyruvate, propionate or other non-aminoacid precursors for gluconeogenesis. Metabolites which are converted toglucose-6-phosphate or ribose-5-phosphate include, but are not limitedto, labeled (²H or ¹³C) hexoses such as [1-²H₁] galactose, [U-¹³C]fructose and the like; labeled (²H or ¹³C) pentoses such as [1-¹³C₁]ribose, [1-²H₁] xylitol and the like, labeled (²H or ¹³C) pentosephosphate pathway metabolites such as [1-²H₁] seduheptalose and thelike, and labeled (²H or ¹³C) amino sugars such as [U-¹³C] glucosamine,[1-²H₁] N-acetyl-glucosamine and the like.

The present invention also encompasses stable isotope labels which labelpurine and pyrimidine bases of DNA through the de novo nucleotidesynthesis pathway. Various building blocks for endogenous purinesynthesis can be used to label purines and they include, but are notlimited to, ¹⁵N-labeled amino acids such as [¹⁵N] glycine, [¹⁵N]glutamine, [¹⁵N] aspartate and the like, ¹³C-labeled precursors such as[1-¹³C₁] glycone, [3-¹³C₁] lactate, [¹³C]HCO₃, [¹³C] methionine and thelike, and ²H-labeled precursors such as ²H₂O. Various building blocksfor endogenous pyrimidine synthesis can be used to label pyrimidines andthey include, but are not limited to, ¹⁵N-labeled amino acids such as[¹⁵N] glutamine and the like, ¹³C-labeled precursors such as [¹³C]HCO₃,[U-¹³ C₄] aspartate and the like, and ²H-labeled precursors (²H₂O).

It is understood by those skilled in the art that in addition to thelist above, other stable isotope labels which are substrates orprecursors for any pathways which result in endogenous labeling of DNAare also encompassed within the scope of the invention. The labelssuitable for use in the present invention are generally conunerciallyavailable or can be synthesized by methods well known in the art.

5.2. Detection of Incorporated Label in DNA

The level of incorporation of stable isotope label into the DNA of cellsis determined by isolating the DNA from a cell population of interestand analyzing for isotope content a chemical portion of the DNA moleculethat is able to incorporate label from an endogenous labeling pathwayusing standard analytical techniques, such as, for example, massspectroscopy, nuclear magnetic resonance, and the like. Methods ofsample preparation will depend on the particular analytical techniquesused to detect the presence of the isotopic label, and will be apparentto those of skill in the art.

In a preferred embodiment of the invention, the presence of the label isdetected by mass spectrometry. For this method of detection, tissue orcells of interest are collected (e.g., via tissue biopsy, blood draw,collection of secretia or excretion from the body, etc.) and the DNAextracted using standard techniques as are well-known in the art. Ofcourse, the actual method of DNA isolation will depend on the particularcell type, and will be readily apparent to those of skill in the art.The cells can be optionally further purified prior to extracting the DNAusing standard techniques, such as, for example, immuno-affinitychromatography, fluorescence-activated cell sorting, elutration,magnetic bead separation, density gradient centrifugation, etc.

If desired, the DNA can then be hydrolyzed to deoxyribonucleosides usingstandard methods of hydrolysis as are well-known in the art. Forexample, the DNA can be hydrolyzed enzymatically, such as for examplewith nucleases or phosphatases, or non-enzymatically with acids, basesor other methods of chemical hydrolysis. Alternatively, prior todetecting the label in the DNA, the DNA incorporating the stable isotopelabel can be detected and measured in intact DNA polymers without beinghydrolyzed to deoxyribonucleosides.

Deoxyribonucleosides are then prepared for mass spectrometric analysisusing standard techniques (e.g., synthesis of trimethylsilyl, methyl,acetyl, etc. derivatives; direct injection for liquid chromatography;direct probe sample introduction, etc.) and the level of incorporationof label into the deoxyribonucleosides determined.

The mass spectrometric analysis is of fragment potentially containingstable isotope label introduced from endogenous labeling pathway. Forexample, the m/z 467-469 fragment of the deoxyadenosine or the m/z 557and 559 fragment of the deoxyguanosine mass spectrum, which contain theintact deoxyribose ring, could be analyzed after [6,6-²H₂] glucoseadministration, using a gas chromatograph/mass spectrometer underelectron impact ionization and selected ion recording mode. Or, the m/z103 and 104 fragment of the deoxyadenosine mass spectrum, which containsthe position C-5 of deoxyribose, could be analyzed after administrationof [6,6-²H] glucose or [6¹³C₁] glucose. In a preferred embodiment, themass spectrometric fragment analyzed is from purine deoxyribonucleosidesrather than pyrimidine deoxyribonucleosides.

The fraction of newly synthesized DNA and therefore newly divided cells(cell proliferation or input rate) or newly removed cells (cell death orexit rate) is then calculated (Table 1).

TABLE 1 abundances abundances m/z m/z dA* f (uncorrected) f (corrected)Day # 457 459 enrichment (% new cells) (% new cells) 1 (Baseline)2844049 518152 0.00000 0.00 0.00 2 1504711 260907 0.00000 0.00 0.00 32479618 453609 0.00298 2.50 3.84 4 3292974 624718 0.00586 4.91 7.55 52503144 461905 0.00451 3.77 5.81 6 1055618 186087 0.00318 2.66 4.09 72186009 394058 0.00193 1.61 2.48

Abundances represent average of three acquisitions. [6,6-²H₂] glucosewas infused intravenously for 48 hrs at 1.25 g/hr to a healthy humansubject with 550 CD4⁺ T cells per mm³ of blood. Plasma glucoseenrichment=11.9%; dA*=deoxyadenosine enrichment based on comparison toabundance corrected standard curve of [5,5-²H₂]deoxyadenosine; funcorrected, calculated as dA enrichment divided by plasma glucoseenrichment; f corrected, calculated as dA enrichment divided by 0.65times plasma glucose enrichment (Macallan et al., 1998 Proc. Natl. Acad.Sci. USA 95:708-713). Calculated cell proliferation rate in totalpopulation: 3.93% per day (21.6 cells/mm³ blood per day). Calculatedremoval (destruction) rate of recently dividing cells: 31.3% per day(half-life=2.2 days).

5.3. Uses

5.3.1. In vitro Uses

In a specific embodiment illustrated by way of example in Section 6,infra, an enrichment of deoxyadenosine (dA) was observed in two celltypes incubated with a stable isotope label and grown as monolayers andin suspension. The dA enrichment correlated closely with the increase incell numbers by direct counting. Therefore, the methods of the inventioncan be used to measure cellular proliferation in a variety ofproliferative assays. For instance, bioassays which use cellularproliferation as a read-out in response to a growth factor, hormone,cytokine or inhibitory factor may be developed by using a stable isotopelabel which targets the de novo nucleotide synthesis pathway. Examplesof such assays include lymphocyte activation by antigen andantigen-presenting cells, apoptosis of target cells induced by tumornecrosis factor and cytotoxicity of tumor cells by cytolyticlymphocytes.

5.3.2. In vivo Uses

Since the methods of the invention using stable isotope labels do notinvolve radioactivity and potentially toxic metabolites, such methodsare particularly useful as a diagnostic tool in measuring cellularproliferation and destruction rates in vivo in subjects, includinghumans. In comparison to conventional methods in humans, the methods ofthe invention are safe, more widely applicable, more easily performed,more sensitive, do not require preservation of cell or tissue anatomyand involve no radioactivity, and produce more accurate results becausethe de novo nucleotide synthesis pathway is constant and predominant, isnot diluted and labels DNA via physiologic substrates rather thanpotentially toxic, non-physiologic metabolites.

A wide variety of medical applications in which cellular proliferationand destruction play an important role are encompassed by the presentinvention. In particular the methods of the invention can be used todetermine the proliferation and destruction rates in cancer, infectiousdiseases, immune and hematologic conditions, organ failure and disordersof bone, muscle and endocrine organs.

5.3.2.1. Cancer Treatments

In one embodiment of the invention, a patient could receive systemic orlocal administration of a stable isotope labeled precursor for the denovo nucleotide synthesis pathway (e.g., [6,6-²H₂] glucose at 1.25 g/hrfor 24-48 hr intravenously) prior to initiation of chemotherapy, andagain 1-2 weeks after starting chemotherapy. A small specimen of theturnover (e.g., by skinny needle aspiration) is performed after eachperiod of stable isotope administration in the synthesis rate of DNA,reflecting inhibition of tumor cell division, could be used as atreatment end-point for selecting the optimal therapy. Typically, thedose of isotope precursor given is enough to allow incorporation intodeoxyribonucleosides above the mass spectrometric detection limits.Samples are taken depending on tracer dilution and cell turnover rates.

5.3.2.2. Cancer Prevention

The risk for breast, colon and other cancers strongly correlates withproliferative stress in the tissue, i.e., hormones, inflammation ordietary factors that alter cell proliferation profoundly affect cancerrates. The ability to characterize a woman's underlying mammary cellproliferative stress and its response to preventative intervention (as,for example, tamoxifen) in early adult life, for example, wouldradically alter breast cancer prevention. The same applies to coloncancer, lung cancer, and other cancers.

5.3.2.3. Aids

Anti-retrovirals in AIDS are intended to block viral replication (abiosynthetic process) in order to reduce CD4⁺ T cell death and turnover.Recent advances in AIDS treatment have focused precisely on thesekinetic processes, although direct kinetic measurements were notavailable. The ability to measure directly these treatment end-pointscan radically change the nature of HIV therapeutics. Physicians canquickly determine whether to begin aggressive anti-retroviral treatmentearly in the disease for each individual patient. In a specificembodiment illustrated by way of example in Section 6, infra, themethods of the invention are used to measure accurately theproliferation and/or destruction rates of CD4⁺ cells in humanimmunodeficiency virus (HIV)-infected patients.

In another specific embodiment illustrated by way of example in Section6, infra, the methods of the invention are used to measure accuratelyand directly the proliferation or destruction rates of T cells,including CD4⁺ and CD8⁺ cells, in vivo in subjects infected with thehuman immunodeficiency virus (HIV). Such methods can be performed priorto, during, or after anti-retroviral treatment of the subject for HIVinfection.

5.3.2.4. Conditions in Which Cellular Proliferation is Involved

A large number of conditions are known to be characterized by alteredcellular proliferation rates and thus can be monitored by methods of theinvention:

Cancer: Malignant tumors of any type (e.g., breast, lung, colon, skin,lymphoma, leukemia, etc.); pre-cancerous conditions (e.g., adenomas,polyps, prostatic hypertrophy, ulcerative colitis, etc.); factorsmodulating risk for common cancers (e.g., estrogens and breastepithelial cells; dietary fat and colonocytes; cigarette smoking oranti-oxidants and bronchial epithelial cells; hormones and prostatecells, etc.). Cells identified above and cells of tissues and organsidentified above are among those cells that are at risk for cancer.

Immune disorders: CD4⁺ and CD8⁺ T lymphocytes in AIDS; T and Blymphocytes in vaccine-unresponsiveness; T cells in autoimmunedisorders; B cells in hypogammaglobulinemias; primary immunodeficiencies(thymocytes); stress-related immune deficiencies (lymphocytes); and thelike.

Hematologic conditions: White blood cell deficiencies (e.g.,granulocytopenia); anemias of any type; myeloproliferative disorders(e.g., polycythemia vera); tissue white cell infiltrative disorders(e.g., pulmonary interstitial eosinophilia, lymphocytic thyroiditis,etc.); lymphoproliferative disorders; monoclonal gammopathies; and thelike.

Organ failure: Alcoholic and viral hepatitis (liver cells); diabeticnephropathy (glomerular or mesangeal cells); myotrophic conditions(myocytes); premature gonadal failure (oocytes, stromal cells of ovary,spermatocytes, Leydig cells, etc.); and the like.

Conditions of bone and muscle: Response to exercise training or physicaltherapy (myocytes or mitochondria in myocytes); osteoporosis(osteoclast, osteoblasts, parathyroid cells) myositis; and the like.

Endocrine conditions: Diabetes (islet β-cells); hypothyroidism andhyperthyroidism (thyroid cells); hyperparathyroidism (parathyroidcells); polycystic ovaries (stromal cells of ovary); and the like.

Infectious diseases: Tuberculosis (monocytes/macrophages); bacterialinfections (granulocytes); abscesses and other localized tissueinfections (granulocytes); viral infections (lymphocytes); diabetes footdisease and gangrene (white cells); and the like.

Vascular disorders: Atherogenesis (smooth muscle proliferation inarterial wall); cardiomyopathies (cardiac myocyte proliferation); andthe like.

Occupational diseases and exposures: Susceptibility to coal dust forblack lung (Coal Worker's Pneumoconiosis) and brown lung (fibroblastproliferative response); susceptibility to skin disorders related to sunor chemical exposures (skin cells); and the like.

The isotope label suitable for use in vivo is prepared in accordancewith conventional methods in the art using a physiologically andclinically acceptable solution. Proper solution is dependent upon theroute of administration chosen. Suitable routes of administration can,for example, include oral, rectal, transmucosal, transcutaneous, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternatively, one can administer a label in a local rather thansystemic manner, for example, via injection of the label directly into aspecific tissue, often in a depot or sustained release formulation.

Determination of a detectable amount of the label is well within thecapabilities of those skilled in the art.

5.4. Methods of Screening an Agent for a Capacity to Induce or InhibitCellular Proliferation

The invention also provides methods for screening an agent or compoundfor a capacity or ability to induce or inhibit cellular proliferation.Such methods comprise contacting a cell with or exposing a cell to asuspected toxic agent or compound. The cell is contacted with adetectable amount of a stable isotope label which is incorporated intoDNA of the cell via the de novo nucleotide synthesis pathway asdescribed herein for other methods of the invention for measuringcellular proliferation and destruction rates. In some such methods, thecell is contacted with or exposed to the agent or compound of interestprior to contacting it with the isotope label. Alternatively, the cellcan be contacted with such agent or compound after it is contacted withthe isotope label. The amount of label incorporated into the DNA,compared to a control application in the cell is not exposed to theagent, indicates the extent of cellular proliferation and therebywhether the agent induces or inhibits cellular proliferation. As withother methods of the invention described supra and infra, the label istypically attached to a precursor of deoxyribose in the de novonucleotide synthesis pathway, the precursor being incorporated intodeoxyribose. In some such methods, as with other methods of theinvention, the precursor is glucose and the label is attached to theglucose. The types of stable isotope labels that can be used with suchmethods are analogous to those described for other methods of theinvention relating to measuring cellular proliferation and destructionrates.

Detection procedures include those well known in the art and thosedescribed in this disclosure regarding methods for measuring cellularproliferation and destruction rates, including mass spectrometry. Aswith other methods of the invention, the DNA is typically—though notnecessarily—hydrolyzed to deoxyribonucleosides prior to detecting thelabel in the DNA. The label can be detected in intact DNA polymers.

The present invention also provides in vivo methods of screening anagent for its capacity or ability to induce or inhibit cellularproliferation in a subject exposed to an agent. Such methods compriseexposing the subject (or a cell of the subject) to the agent, andadministering a detectable amount of a stable isotope label to thesubject. The label is incorporated into DNA of the subject via the denovo nucleotide synthesis pathway. The label incorporated into the DNAof a cell of interest in the subject is detected to determine the degreeof cellular proliferation of the cell of interest in the subject. Theamount of label detected in the DNA—relative to a control application inwhich the same subject is not exposed to the agent or compared to acontrol group of comparable subjects not exposed to the agent—indicatesthe extent of cellular proliferation and thus whether the agent inducesor inhibits cellular proliferation in the subject. The manner ofadministration, type of stable isotope label, and method of detectionare analogous to those described for other methods of the inventionrelating to measuring cellular proliferation and destruction rates.

With these screening methods, the capacity of an agent to directlyinduce or inhibit cellular proliferation of a cell can be determined bydirectly exposing the cell of interest to the agent and then measuringthe proliferation of the cell.

The invention also provides methods of screening an agent for a capacityto indirectly induce or inhibit cellular proliferation; such methodscomprise exposing one cell or a population of dividing cells of thesubject to the agent and monitoring the rate of cellular proliferationof a second cell or second population of cells different from the firstcell. For example, the cell that is directly exposed to the agent can befrom one tissue of the subject, while the cell of interest can be from asecond tissue. Alternatively, the cell that is directly exposed to theagent can comprise a first type of cell line, while the cell of interestcomprises a second type of cell line which is different from the firstcell line. The capacity of the agent to induce or inhibit cellularproliferation indirectly in a second cell of interest is determined bydetecting the incorporation of the label into the DNA of the secondcell. In such methods, cellular proliferation of the second cell istypically mediated by contact or association of the second cell with thefirst cell—or product of the first cell—which has been exposed to theagent.

With screening methods of the invention, cellular proliferation can bemeasured in vitro and in vivo in animals and humans as described indetail herein for methods of the invention for measuring cellularproliferation. The compound or agent can be administered, for example,to a cell or tissue in vitro or to an organism in vivo, followed bymeasurement of cellular proliferation.

In some screening methods of the invention, the label can beincorporated into a precursor of deoxyribose, and the label can comprisea labeled glucose, as for other methods of the invention for measuringcellular proliferation described throughout this application. Inaddition, in some such screening methods, the DNA is hydrolyzed todeoxyribonucleosides and the label is detected by mass spectrometry.Furthermore, as with other methods for measuring cellular proliferationdescribed supra and infra, the DNA can be extracted from a variety ofcells, including cells particularly at risk for cancer (e.g, breast,colon, or bronchial epithelial cells), lymphocytes, CD4⁺ T cells, orCD8⁺ T cells.

Screening methods of the invention can employ either a non-radioactivestable isotope label or a radioactive label, including those describedherein that are employed with methods for measuring cellularproliferation. Non-radioactive stable isotope labels are particularlyadvantageous because they are non-toxic and thus safe for use in animalsand humans, as described in detail supra and infra in this disclosure.

The screening methods of the present invention can be used to test awide variety of compounds and agents for their respective abilities toinduce cellular proliferation. Such compounds and agents include, butare not limited to, for example, carcinogens, suspected toxic agents,chemical compounds, mutagenic agents, pharmaceuticals, foods, inhaledparticulates, solvents, particulates, gases, and noxious compounds insmoke (including cigarette and cigar smoke, and smoke produced byindustrial processes), food additives, solvents, biochemical materials,hormones, drugs, pesticides, ground-water toxins, environmentalpollutants, proliferogens which stimulate cellular proliferation, andany other compounds or agents that are known or suspected to increasethe risk of cancer. Agents which can be screened for their capacity orability to cause cellular proliferation also include, but are notlimited to, for example, radon, microwave radiation, electromagneticradiation, electromagnetic fields, radiation produced by cellulartelephones, heat, and hazardous materials and conditions produced orpresent in industrial or occupational environments.

With screening methods of the invention, the proliferation rates ofcells that have not been exposed to agents or compounds of interest canbe compared to the proliferation rates of cells that have been exposedto the agents or compounds of interest. In addition, such screeningmethods can be used to compare the rate of cellular proliferation in aparticular cell of interest before and after exposure to a specificagent or compound of interest, including those described herein.

As described supra, the isotope label suitable for use in vivo isprepared in accordance with conventional methods in the art using aphysiologically and clinically acceptable solution. Proper solution isdependent upon the route of administration chosen. Suitable routes ofadministration can, for example, include oral, rectal, transmucosal,transcutaneous, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternatively, one can administer a label in a local rather thansystemic manner, for example, via injection of the label directly into aspecific tissue, often in a depot or sustained release formulation.

Determination of a detectable amount of the label is well within thecapabilities of those skilled in the art.

5.5. Methods for Determining Susceptibility and Risks of a Subject toDiseases Which Induce or Inhibit Cellular Proliferation

The present invention also provides methods for assessing or measuringthe susceptibility of a subject, including animals and humans, to adisease which induces or inhibits cellular proliferation. Such methodscomprise exposing the subject to a condition or an agent which causes orstimulates the disease and measuring the rate of cellular proliferationin the subject by the in vivo methods for measuring cellularproliferation in a cell of interest in the subject as described hereinand throughout this application. Methods for measuring cellularproliferation in a subject comprise, for example, administering adetectable amount of a stable isotope label to the subject. The label isincorporated into DNA of the subject via the de novo nucleotidesynthesis pathway. The label in the DNA of a cell of interest in thesubject is detected to determine cellular proliferation in the cell ofinterest. See methods for measuring cellular proliferation described indetail throughout this application.

5.6. Individualization of Medical Risk Assessment for a Disease:Personalized Risk Assessment

In another aspect, the present invention provides methods fordetermining an individual's personal risk for acquiring a particulardisease or medical condition that involves or induces cellularproliferation or cellular destruction. It is well known that individualsdiffer not only in their exposure to disease risk factors, but also intheir susceptibility to risk factors. Current public healthrecommendations regarding risk reduction are generally collective ratherthan truly personalized; that is, an individual is classified accordingto known epidemiologic variables (as, for example, a post-menopausalCaucasian female of Northern European ethnic background with low bodyweight and two pregnancies), and a statistical risk for a particularcondition (such as breast cancer, endometrial cancer, osteoporosis,etc.) is estimated. Decisions regarding disease risk and potentialbenefits of preventative measures (such as using tamoxifen to reducebreast cancer risk) would ideally be personalized rather than based onstatistical risks.

In one embodiment, the present invention provides methods fordetermining the susceptibility of a subject to a disease or disorderwhich changes or alters the rate of cellular proliferation and/orcellular destruction (e.g., induces or inhibits the rate of cellularproliferation and/or cellular destruction) in the subject. Such methodscomprise exposing the subject to a condition or an agent which canproduce the disease or disorder, administering a detectable amount of astable isotope label to the subject, which label is incorporated intoDNA of the subject via the de novo nucleotide synthesis pathway, anddetecting the label in the DNA of the subject. The subject can beexposed to the agent or condition producing the disease or disorder insuch a manner that the subject acquires only a transient or mild form ofthe disease. For example, the agent can be administered in a low dosagewhich produces only a mild form of the disease or disorder. Where thedisease or disorder induces cellular proliferation, an increase in labelin the DNA of the subject—compared to a control application in which thesubject is not exposed to the condition or agent or compared to acontrol group of comparable subjects not exposed to the condition oragent—indicates an increase in the rate of cellular proliferation andevidences the individual subject's susceptibility to a disease ordisorder. Such information provides a prediction of the subject'ssusceptibility to that disease or disorder. Correspondingly, the rate ofloss or decay of label in the DNA of the subject indicates a change inthe rate of cellular destruction. Where the disease or disorder inducescellular destruction, a loss of label in the DNA of the subject (e.g., arapid loss)—compared to a control application in which the subject isnot exposed to the condition or agent or compared to a control group ofother comparable subjects not exposed to the condition oragent—demonstrates an increase in the rate of cellular destruction andsusceptibility of the subject to that disease. Such information providesa prediction of the subject's risk for such disease or disorder whichincreases the rate of cellular destruction.

Such methods allow for personalization of risk assessment of acquiring adisease or condition involving altered rates of cellular proliferationor destruction by measurement of the actual rates of cell proliferationand/or destruction in an individual subject. These methods are usefulbecause they permit an individual to make important decisions regardinghis or her lifestyle (e.g., diet, occupation, habits, medications, etc.)and/or medical interventions (e.g., pharmaceuticals, hormones, vitamins,etc.). By measuring an individual's specific susceptibility to a diseasewhich involves altered rates of cellular proliferation or destruction,decisions pertaining to one's lifestyle or medical treatments can bebased on an individual subject's actual risk of acquiring a particulardisease—rather than being based on collective risk inferred statisticsfor a general population of individuals. With such methods, theeffectiveness of a medical intervention, life-style intervention, orother intervention in an individual can also be directly measured ratherthan assumed (e.g., to ascertain whether an intervention, such astamoxifen therapy, is, in fact, successfully reducing proliferation ofbreast epithelial cells in a particular female subject at high risk forbreast cancer).

Medical risk assessments for a wide variety of diseases and conditions,including those described in Section 5, supra, and Section 6, infra, canbe made. By way of example, such methods of the invention are useful inassessing a particular individual's risk of acquiring Black Lung orBrown Lung disease—an occupational hazard for workers in coal mines. Oneof the classic observations regarding Coal Worker's Pneumoconiosis(Black Lung) is that inter-individual variability in susceptibilityexists among coal workers (Balaan et al., 1993, Occup. Med. 8(1):19-34;Borm et al., 1992, Toxicol. Lett. 64/65:767-772; Liddell and Miller,1983, Scand. J. Work Environ. Health. 9:1-8; Katsnelson et al., 1986,Environ. Health Perspect. 68:175-185. The rate at which pulmonarychanges occur in individuals exposed to conditions that can precipitatethis disease varies tremendously—with some people developing onlymoderate coughing and sputum production after 10 years of dust-exposure,while other people rapidly develop fibrotic lungs, severe shortness ofbreath, and low blood oxygen.

Some factors influencing the rate of Black Lung disease progression areknown (e.g., cigarette smoking (Balaan et al., 1993, Occup. Med.8(1):19-341)), but it is not currently possible to identify highlysusceptible individuals. Some investigators have emphasized theimportance of early identification of those workers having accelerateddeclines in pulmonary function and relocation of such workers from theworkplace (Balaan et al., 1993, Occup. Med. 8(1):19-341). Identificationof susceptible individuals is the ideal preventative strategy for anypublic health problem, short of removing the inciting agent itself. Itis believed that environmental exposure to the agent causing Black Lungdisease or related conditions and individual susceptibility (based ongenetics, nutritional status, co-factors, etc.) to Black Lung diseaseare both required to produce the disease. Black Lung disease or relatedconditions causes fibrogenesis (lung scarring).

The final common pathway leading to fibrotic lungs for all individualsis the activation of cells responsible for scarring (fibroblasts) todivide and to produce the protein comprising scars (collagen). Such lungdamage can be measured directly in at-risk humans using the methods ofthe invention. Since the proliferation of fibroblasts represents a cellproliferation process, this pathogenesis is ideally suited forobservation using the in vivo methods of the present invention formeasuring cellular proliferation. The methods of the present inventionare especially useful in this regard because they allow a clinician orresearcher to measure cellular proliferation processes precisely anddirectly in an individual (e.g., coal worker)—rather than looking forindirect signs of developing fibrogenesis and scarring and/or waitingfor irreversible scarring to be manifested by x-ray or functionalchanges.

The methods for determining a subject's risk or susceptibility to aparticular disease or condition involving cellular proliferationtypically comprise exposing the subject to an agent or condition whichproduces, induces, or stimulates the disease and measuring the rate ofcellular proliferation in the subject by the in vivo methods formeasuring cellular proliferation described throughout this application.An assessment of a subject's susceptibility or risk of acquiring BlackLung disease can be determined, for example, by administering orally tothe subject a marker nutrient solution (i.e., a solution containing alabeled compound which is ultimately incorporated into the DNA of theindividual, as for other cellular proliferation measurement methodsdescribed herein), and then collecting the sputum (or lung washings).The label incorporated into the DNA is then detected. This procedure canbe performed on a subject both before occupational exposure (i.e.,before starting to work in a coal mine) and after such exposure for asuitable period of time (e.g., six months). The presence of rapidlyproliferating fibroblasts (Hellerstein and Neese, 1992, Am. J. Physiol.263:E988-E1001) or newly synthesized collagen (Hellerstein and Neese,1992, Am. J. Physiol. 263:E988-E1001; Papageorgopoulos et al., 1993,FASEB J. 7(3):A177; Caldwell et al., 1993, Am. Soc. Mass Spectrom. Conf.p. 7) indicates whether fibrogenesis and tissue scarring were activelyoccurring before permanent and irreversible damage had developed.

The ability to measure fibroblast proliferation in a individual's lungsdirectly is useful in determining the susceptibility of the individualto Black Lung disease. Such measurements is also extremely useful inmonitoring or ascertaining the effectiveness of standard treatmenttherapies in patients suffering from diseases such as Black Lung diseaseor evaluating the effectiveness of new treatment therapies (e.g.,anti-oxidants, anti-fibrogenic factors, cytokine blockers, etc. (Lappand Castranova, 1993, Occup. Med. 8(1):35-56)) for such diseases. Suchmethods of the invention offer distinct advantages over currentlyemployed methods; for example, with such methods, it is not necessary towait for irreversible x-ray changes or loss of pulmonary function todevelop before adjustments are made to an individual's treatmenttherapy. Early preemptive measures rather than after-the-fact responsescan be determined and implemented.

The information resulting from such methods would allow medicalprofessionals to provide guidance to individuals that are resistant tothe disease or condition as well as to individuals that are susceptibleto the disease. Individuals that are not susceptible to the disease orcondition might be advised to continue to work in the environmentwithout fear of acquiring the disease, while disease-sensitiveindividuals might be counseled to changing jobs or try medicalinterventions that might reduce or prevent lung damage (see text above).

5.7. Radioactive Isotope Labels for Use in Methods for MeasuringCellular Proliferation

The present invention also provides methods for measuring cellularproliferation and destruction rates which employ non-stable radioactiveisotope labels to endogenously label DNA through the de novo nucleotidesynthesis pathway in a cell. Such methods comprise contacting a cellwith a detectable amount of a radioactive isotope label which isincorporated into DNA via the de novo nucleotide synthesis pathway, asdescribed previously for methods utilizing non-radioactive stableisotopes. The radioactive isotope label is then detected in the DNA tomeasure the rate of cellular proliferation or destruction.

Methods utilizing radioactive isotope labels offer particular advantagesand uses because such labels and the techniques for detecting suchlabels are often less expensive than non-radioactive stable isotopelabels and the corresponding techniques for detecting stable isotopelabels. For example, radioactivity measurement techniques for detectingradioactive labels are typically much less costly to perform than arethe standard mass spectrometric techniques utilized for detecting stableisotope labels. The invention also provides methods for measuringcellular proliferation in a proliferating or dividing population ofcells that are dividing and producing progeny which employ bothradioactive isotope labels and stable isotopes to endogenously label DNAthrough the de novo nucleotide synthesis pathway. Such methods comprisecontacting the proliferating population of cells with a detectableamount of a stable isotope label which is incorporated into DNA via thede novo nucleotide synthesis pathway. The stable isotope labelincorporated into the DNA is detected to determine the rate of cellularproliferation in the population of cells by techniques described herein,including mass spectrometric techniques. The proliferating population ofcells is also contacted with a detectable amount of a radioactiveisotope label, which incorporates into DNA via the de novo nucleotidesynthesis pathway. The radioactive isotope label incorporated into theDNA is detected by standard radioactivity counting techniques to measurecellular proliferation in the proliferating population of cells. Theproliferating population of cells can be contacted first with either thestable isotope label or the radioactive isotope label and, followingincorporation of such label into the DNA, the amount of such label inthe DNA can be measured and determined by the detection proceduresdescribed herein. Alternatively, in some such methods, the population ofcells can be contacted simultaneously with the stable and radioactiveisotope labels, and the detection of both such isotopes can be performedsimultaneously.

Methods of the invention utilizing both non-radioactive stable isotopelabels and radioactive isotope labels are useful for performingdouble-labeling studies and for measuring cellular proliferation ratesover time—even a short time (such as, e.g., several minutes or hours).Notably, because different techniques are typically used to detectradioactive isotope labels and non-radioactive stable isotope labels,the amount of each type of label incorporated into the DNA can bemeasured independently—without risk that the measurement of one type oflabel might interfere with the measurement of the other type of label.

With some methods for measuring cellular proliferation rates in adividing population of cells using both stable and radioactive isotopelabels, the stable and radioactive isotope labels are each attached to aprecursor of deoxyribose in the de novo nucleotide synthesis pathway.Each precursor is then incorporated into deoxyribose of the DNA. In apreferred embodiment, the stable isotope and the radioactive isotopelabel each comprise a labeled glucose.

By way of example, in one embodiment, a baseline measurement of cellularproliferation is first performed after contacting a cell with adetectable amount of a stable isotope label which is incorporated intothe DNA via the de novo nucleotide synthesis pathway. Measurement ofcellular proliferation is then repeated after contacting the cell with aradioactive isotope label which is also incorporated into the DNA viathe de novo nucleotide synthesis pathway. In this way, a change in therate of cellular proliferation over time is determined withoutinterference or carry-over from the initial stable isotope labeledmaterial. Furthermore, the second cellular proliferation measurementusing radioactive isotope label can be conducted shortly after the firstcellular proliferation measurement without waiting for the stableisotope label to be removed from or washed out of the system, becausethere is no risk that the stable isotope label with interfere with anaccurate measurement of the radioactive label. The DNA incorporating thestable isotope label and/or the DNA incorporating the radioactiveisotope label can be hydrolyzed to deoxyribonucleosides prior todetecting the label in the DNA or can be detected and measured in intactDNA polymers.

The cellular proliferation rates of a variety of proliferatingpopulations of cells, including cancer cells and lymphocytes (e.g., CD4⁺and CD8 ⁺ cells), etc., can be measured by these methods.

In methods of the invention employing radioactive isotope labels,incorporation of the radioactive label into the DNA of a cell can bemeasured by a variety of well-known techniques, including radioactivitymeasurement techniques, such as liquid scintillation counting or gammacounting, and accelerator mass spectrometry. Accelerator massspectrometry is particularly useful in measuring incorporation ofcertain radioactive labels (such as ¹⁴C) into cellular DNA. Notably,accelerator mass spectrometry cannot be used to measure theincorporation of stable isotope labels (e.g., ¹³C incorporation) intocellular DNA. Although accelerator mass spectrometry is typicallyexpensive, it allows for detection of extremely low levels ofradio-isotope incorporation (in particular, ¹⁴C incorporation) intocellular DNA. Given that extremely small amounts of a radioactive label(e.g., ¹⁴C) in DNA can be detected by this technique, only small amountsof radioactive label need be used, thereby lessening or eliminatingpotential toxicities associated with such a label. In methods employingboth radioactive and stable isotope labels, the stable isotope label canbe detected by standard well-known techniques, including massspectrometry, as described for other methods of the present invention.

Radioactive isotope labels suitable for use with methods of theinvention are known to those of ordinary skill in the art. Examplesinclude the tritiated thymidine (³H-dT) and bromodeoxyuridine (BrdU)(Waldman et al., 1991, Modern Pathol. 4:718-722; Gratzner, 1982, Science218:474-475). Such radioactive isotope labels can be prepared asdescribed supra for the stable isotope labels in accordance withconventional methods in the art using a physiologically and clinicallyacceptable solution. Proper solution is dependent upon the route ofadministration chosen.

Procedures for labeling a precursor of DNA, such as deoxyribose, with aradioactive isotope label and incorporating such radioactive isotopelabel into DNA via the de novo nucleotide pathway are analogous to thosedescribed herein regarding methods for measuring cellular proliferationand destruction and as will be apparent to those of skill in the artbased on the detailed disclosure provided herein.

Determination of a detectable amount of either the radioactive isotopeor stable isotope label is well within the capabilities of those skilledin the art.

The present invention is further illustrated by the following examples.These examples are merely to illustrate aspects of the present inventionand are not intended as limitations of this invention.

6. EXAMPLES Measurement of Cell Proliferation by Labeling DNA withStable Isotope-Labeled Glucose

6.1. Materials and Methods

6.1.1. Isolation of Deoxyribonucleosides from DNA

DNA was prepared from cells or tissues by phenol-chloroform-isoamylalcohol extraction of cell suspensions or tissue homogenates. Yield andpurity were confirmed by optical density. After heat denaturation, DNAwas hydrolyzed enzymatically to deoxyribonucleosides by sequentialdigestion with nuclease P1, phosphodiesterase, and alkaline phosphatase,as described by Crain et al. (Crain, 1990, Methods Enzymol.193:782-790). Nucleoside yield and purity were confirmed by HPLC using aC-18 column and water-methanol gradient (Shigenaga et al., 1994, MethodsEnzymol. 234:16-33).

6.1.2. Deruvatization of Deoxyribonucleosides and Analysis by GasChromatography Mass Spectrometry (GC-MS)

Trimethylsilyl derivatives of nucleosides were synthesized by incubationof lyophilized hydrolysates with BSTFA:pyridine (4:1) at 100° C. for 1hour. Samples were analyzed by GC-MS (DB-17 HT column, J&W Scientific,Folsom Calif.; HP 5890 GC and 5971MS, Hewlett Packard, Palo Alto,Calif.). Abundances of ions at mass to charge ratio (m/z) 467 and 469were quantified under selected ion recording mode for deoxyadenosine(dA); and m/z 555 and 557 were monitored for deoxyguanosine (dG). Underthe derivatization and GC-MS conditions employed, the purines (dA anddG) gave larger peaks (FIG. 3) than the pyrimidines, resulting ingreater sensitivity and a higher signal-to-noise ratio. To measure theenrichment of glucose, plasma or culture media were deproteinized withperchloric acid and passed through anion and cation exchange columns(Neese et al., 1995, J. Biol. Chem. 270:14452-14463). The glucosepenta-acetate derivative was formed by incubation with acetic anhydridein pyridine. GC-MS analysis was performed as described previously (Neeseet al., 1995, J. Biol. Chem 270:14552-14663), monitoring m/z 331 and 333under selected ion recording.

6.1.3. In vitro Studies

Initial studies of label incorporation from [6,6-²H₂] glucose intocellular DNA were performed in tissue culture cell lines. Two cell lineswere used: a hepatocyte cell line, HepG2, and a lymphocytic cell line,H9, which is a CD4⁺ T cell line. HepG2 cells were grown in 10 ml disheswith alpha-modified Dulbecco's minimum essential media (MEM). H9 cellswere grown in suspension in RPMI 1640. Both were grown in the presenceof 10% dialyzed fetal calf serum and antibiotics (all reagents wereobtained from Gibco-BRL, Gaithersburg, Md., except where stated). Inboth cases the number of cells present was measured by counting analiquot on a Coulter ZM0901 cell counter. For HepG2 cells, platingefficiency was corrected for by counting an identical plate at thebeginning of each labeling phase. Cells were labeled by addition of[6,6-²H₂] glucose (Cambridge Isotope Laboratories, Andover, Mass.) suchthat labeled glucose constituted 10-20% by weight of total glucosepresent (100 mg/L for MEM-a and 200 mg/L for RPMI 1640). In someexperiments, glucose free medium was used and only 100% labeled glucosewas present in the medium. Additional experiments were carried out inthe presence of [U-¹³C₆] glucose and [2-¹³C₁] glycerol (CambridgeIsotope Laboratories, Andover, Mass.).

6.1.4. Animal Studies

Four rats (approximately 250 g) were infused with labeled glucose.Intravenous canulae were placed under anesthesia (Hellerstein et al.,1986, Proc. Natl. Acad. Sci. USA 83:7044-7048). After a 24-48 hrrecovery period, [6,6-²H₂] glucose was infused as a sterile 46 mg/mlsolution at 0.5 ml/hr for approximately 24 hours. Food was withdrawn atthe beginning of the isotope infusion. This dose was expected to achieveaverage plasma glucose enrichments of about 10%, based on previousstudies in fasting rats (Neese et al., 1995, J. Biol. Chem.270:14452-14463). At the end of the infusion period, animals weresacrificed. Blood for plasma glucose enrichment and tissues for DNAextraction were collected and frozen prior to analysis. A section of theintestine approximately 30 cm in length was excised from just below theduodenum in each rat. The intestinal segments were everted and washed.Epithelial cells were released from the submucosa by incubation withshaking in buffer containing 5 mM EDTA at 37° C. for 10 min, asdescribed by Traber et al. (Traber et al., 1991, Am. J. Phvsiol.260:G895-G903). DNA was extracted from cell preparations, thenhydrolyzed to nucleosides and analyzed by GC-MS (FIG. 2).

6.1.5. Studies of Granulocyte Kinetics in Human Subjects

In order to investigate the application of the methods of the inventionin clinical settings, four volunteers received intravenous infusion of[6,6-²H₂] glucose (60 g over 48 h) in the General Clinical ResearchCenter at San Francisco General Hospital. One subject was a healthynormal volunteer and the other three were HIV-seropositive men, who wereparticipating in lymphocyte kinetic studies (blood CD4 T cell counts inthe range 215-377/mm³). None had a clinically apparent infection at thetime of the infusion. In order to enable high and relatively constantenrichments of plasma glucose and maximize labeling of cellular DNA,dietary carbohydrate was restricted (mean intake 46 g/day) during the2-day period of the infusion. A heparinized blood sample was drawn atbaseline and every 12 hr during the infusion, for estimation of plasmaglucose enrichment. After the 48-hr infusion, blood was collected dailyfor 10 days and granulocytes and mononuclear cells were separated bygradient centrifugation (Vacutainer CPT, Becton Dickinson, FranklinLakes, N.J.). Granulocyte DNA was extracted, hydrolyzed to nucleosidesand analyzed by GC-MS, as described in Section 6.1.2, supra. Allprocedures received prior approval by the University of California atSan Francisco Committee on Human Research and the University ofCalifornia at Berkeley Committee for the Protection of Human Subjects,and written informed consent was obtained from subject for allprocedures carried out.

6.2. Results

6.2.1. Development of Analytic Method

Derivatization is required to volatilize deoxyribonucleosides for GC-MSanalysis (Blau and Halket, 1993, Handbook of Derivatives forChromatography 2nd ed.). The highest abundances with TMS derivatizationwas observed, compared to methylation or acetylation. The GC-MS scans ofa typical TMS-derivatized sample, analyzed under electron impactionization, are shown (FIG. 3). dA and dG eluted from the GC columnshowed well defined peaks. As described previously (McCloskey, 1990,Methods Enzymol. 193:825-841), the dominant ions in the spectra werefrom the base moiety that were unlabeled from [6,6-²H₂] glucose. Theparent ions, m/z 467 and 557 for dA-TMS₃ and dG-TMS₄, respectively, werewell represented and were present in a region of the mass spectrum withlittle background. Labeled samples contained an excess of the M+2 ions469 and 557; the ratios of 469 to 467 and 557 to 559 were used forquantification.

Abundance sensitivity of isotope ratios (concentration dependance) wasobserved for dA and dG, as described for GC-MS (Neese et al., 1995, J.Biol. Chem. 270:14452-14463; Patterson and Wolfe, 1993, Biol. MassSpectrom. 22:481-486). Samples were therefore always analyzed atabundances matched to those in the standards used for baseline (naturalabundance) subtraction, when calculating isotope enrichments. Inenriched samples, the measured enrichments of dA were not significantlydifferent from dG, as expected. Only data from dA are shown below.

6.2.2. In vitro Cell Proliferation Studies

The enrichment of dA derived from cells grown in media containing 10-15%[6,6-²H₂] glucose increased progressively with time (FIGS. 4A and 4B).This was demonstrated for both a hepatocyte cell line (HepG2) grown asmonolayers on plates, and for a T-lymphocytic cell line (H9) grown insuspension. When compared to the number of cells measured by directcounting, dA enrichment correlated closely with the increase in cells bydirect counting (FIGS. 4C and D). The correlation coefficient betweenthe fraction of new DNA (calculated from the ratio of M2 enrichments indA to medium glucose) and the percentage of new cells by direct countingwas 0.984 with HepG2 cells and 0.972 for H9 cells.

The enrichment of the true intracellular dATP precursor pool for DNAsynthesis using growing cells was equal in theory to the dA enrichmentin DNA at 100% new cells (i.e., when only labeled DNA was present).Extrapolation of the labeling time course experiments to 100% new cellsgave estimated plateau dA enrichments of 0.725 of the medium glucoseenrichment for HepG2 cells and 0.525 for H9 cells (FIGS. 4A-4D).

In order to test directly the relationship between enrichments ofextracellular glucose and intracellular DNA precursors, cells were grownfor prolonged periods in medium containing 100% [6,6-²H₂] glucose withrepeated replating or subculture of cells, for a total of 53 days forHepG2 cells and 25 days for H9 cells. At the end of the experiment,<0.1% of DNA present could be accounted for by the initial unlabeledcells. Maximum enrichment of dA was about 65% for both HepG2 and H9cells (FIGS. 5A and B). One possible explanation for this dilution ofextracellular labeled glucose could be the synthesis of glucose withinthe cell, e.g., from gluconeogenesis (GNG), since unlabeled amino acidprecursors for GNG were present in the culture medium. Alternatively,some exchange of the label might have occurred during intracellularmetabolism of glucose, either during glycolysis and passage through thetricarboxylic acid cycle or during the non-oxidative portion of thepentose-phosphate pathway (Wood et al., 1963, Biochemische Zeitschrift338:809-847).

If intracellular unlabeled glucose from GNG were the dominant origin ofdilution, dA from H9 cells might approach closer than the Hep G2 cellsto 100% of medium glucose enrichment. However, this was not found to bethe case (FIGS. 4A-4D, 5A and 5B). A more direct test would be theincorporation of GNG precursors into dA in DNA by HepG2 cells. In orderto test this hypothesis, both HepG2 and H9 were cultured in the presenceof [2-¹³C₁] glycerol. By applying the theory of combinatorialprobabilities, or the mass isotopomer distribution analysis (MIDA)technique (Neese et al., 1995, J. Biol. Chem. 270:14452-14463;Hellerstein et al., 1992, Am. J. Physiol. 263:E988-E1001), the fractionof deoxyribose in dA that came from GNG could then be calculated. WhenHepG2 cells were grown in media to which [2-¹³C₁] glycerol had beenadded at concentration of 20 μg/ml, negligible incorporation of ¹³C intodA was found. In the presence of 100 μg/ml [2-¹³C₁] glycerol(approximately 2-3 times plasma glycerol concentrations), enrichment ofboth M₊₁ and M₊₂ ions was observed in dA. Applying MIDA revealed that17.8% of dA pentose ring synthesis appeared to arise from GNG ratherthan utilization of extracellular glucose. H9 cells grown in thepresence of 100 μg/ml [2-¹³C₁] glycerol revealed no measurable GNG, asexpected.

Duplicate pairs of cell cultures were also grown in the presence of 10%[6,6-²H₂] glucose with and without the addition of unlabeled glycerol(100 g/ml). Such unlabeled glycerol did not affect labeling in H9 cells;in HepG2 cells, incorporation into dA was reduced by 7%. Thus, itappears that the availability of GNG precursors had only a small effecton the labeling of DNA in cells capable of GNG and GNG does not fullyexplain the intracellular dilution of dA.

If the roughly 35% dilution between extracellular glucose and dA in DNAwere due to exchange of ²H for ¹H in intracellular glucose cycles,carbon label in [U-¹³C₆] glucose should undergo re-arrangement.Accordingly, HepG2 and H9 cells were grown in the presence of 10%[U-¹³C₆] glucose. If there were no metabolism through pathways such asthe non-oxidative portion of the pentose phosphate pathway, the dNTP'sfrom this precursor should retain all five labeled carbons and have amass of M₅. The M₅ enrichment increased in a similar fashion to thatobserved with the M₂ ion from [6,6-²H₂] glucose. An asymptote ofapproximately 80% of the extracellular enrichment was reached in HepG2cells (FIGS. 6A and 6B) while in H9 cells the asymptote wasapproximately 60% of extracellular glucose enrichment. When the M₀ to M₅spectrum was analyzed, enrichments of M₂, M₃, and M₄ ions were seen inaddition to the expected enrichment of M₅. This phenomenon was observedin both H9 and HepG2 cells, although the relative abundance of theseions was greater from H9 cells.

The above cell culture experiments were performed in the absence ofdeoxyribonucleosides in the medium. Previous studies with lymphocytecell lines (Reichard, 1978, Fed. Proc. 37:9-14; Reichard, 1988, Ann.Rev. Biochem. 57:349-374) have suggested that increasing theavailability of extracellular deoxyribonucleosides does not reduce, andmay even increase, activity of ribonucleoside reductase and theendogenous synthesis pathway for purine dNTP's (FIG. 1). To testdirectly the effects of increased availability of extracellulardeoxyribonucleosides, HepG2 and H9 cells were grown in the presence ofan equimolar mixture of the four deoxyribonucleosides. Twoconcentrations, 20 and 100 μM, were chosen to reproduce or exceed thoseprevailing in tissues; plasma concentrations are normally of the orderof 1 μM and tissue concentrations may range between 1 and 100 μM (Cohenet al., 1983, J. Biol. Chem. 258:12334-12340). Six flasks of H9 cellswere grown in parallel in media labeled with ca. 10% [6,6-²H₂] glucose(Table 2).

TABLE 2 Effect of extracellular deoxyribonucleosides on incorporation of[6,6-²H₂] glucose into dA in DNA Extracellular DeoxyribonucleosideConcentration (μM) 0 0 20 20 100 100 Lymphocytes (H9) dA/glucose ratio0.527 0.522 0.535 0.528 0.534 0.514 Fraction new cells (by counting)0.849 0.851 0.867 0.856 0.839 0.822 Extrapolated dA/glucose (100% newcells) 0.620 0.613 0.617 0.617 0.637 0.626 Hepatocytes (HepG2)dA/glucose ratio 0.386 0.385 0.381 0.370 0.339 0.344 Fraction new cells(by counting) 0.568 0.589 0.536 0.565 0.626 0.570 ExtrapolateddA/glucose (100% new cells) 0.680 0.653 0.711 0.655 0.541 0.603

Two were grown without added deoxyribonucleosides, two were grown at thelower and two at the higher deoxyribonucleoside concentrations. After 90hours, 85% of cells were new by counting. The experiment was alsoperformed with HepG2 cells, yielding an average increase in cell numberrepresenting about 58% new cells. In H9 cells the presence ofextracellular deoxyribonucleosides at either 20 or 100 μM did not reducethe incorporation of label from glucose into dA and thus did not appearto suppress the activity of the de novo nucleotide synthesis pathway. InHepG2 cells there was no appreciable reduction in incorporation at 20μM, although there was a small (ca. 12%) reduction at 100 μM. In H9cells, the extrapolated ratio of dA/glucose at 100% new cells (based oncell counting) was reproducibly between 62-64%. For HepG2 cells, theratio ranged between 54-71%.

6.2.3. In vivo Labeling of DNA: Animal Studies

In rats (n=4) receiving intravenous infusion of [6,6-²H₂] glucose,plasma glucose enrichment at sacrifice was 13.2±0.9%. The mean plasmaglucose enrichment for the whole 24 hour infusion period was less thanthis value because plasma glucose enrichment progressively increasedduring fasting, as the Ra glucose progressively fell (Rocha et al.,1990, Eur. J. Immunol. 20:1697-1708). The mean plasma glucose enrichmentwas estimated from two rats receiving labeled glucose infusion, and inwhich repeated blood samples were taken via arterial blood-drawing line.The mean enrichment for the 24 hr fasting period was 0.70 of the finalenrichment; accordingly, this value was used for calculating the meanglucose enrichments for the four experimental rats (9.2±0.6%).

Differing enrichments were found in dA from the three tissues studied(Table 3).

TABLE 3 In vivo incorporation of [6,6-²H₂] glucose into dA in DNA invarious tissues in rats dA Enrichment Percent New Cells Turnover time(d) Rate Constant, K(d⁻¹ ) t_(1/2) (d) Tissue (%) Uncorrected CorrectedUncorrected Corrected Uncorrected Corrected Uncorrected CorrectedIntestinal 3.18 ± 0.24 34.6 ± 4.2 53.2 ± 6.5 2.83 ± 0.37 1.84 ± 0.24 — —— — epithelium Thymus 2.33 ± 0.08 25.3 ± 2.2 38.9 ± 3.1 — — 0.302 ±0.030  0.51 ± 0.058 2.31 ± 0.23 1.36 ± 0.15 Liver 0.25 ± 0.06  2.7 ± 0.5 4.2 ± 0.9 — — 0.028 ± 0.005 0.044 ± 0.008  25.4 ± 4.9  16.2 ± 3.1  Meanplasma glucose enrichment was 9.2 ± 0.6% and mean duration of infusionwas 23.2 ± 0.1 hr. Uncorrected calculations use plasma glucoseenrichments as precursor for DNA synthesis; corrected calculations use acorrection factor of 0.65 × plasma glucose enrichment to account fordilution in the intracellular precursor pool (see text). A linearkinetic model was used for intestinal epithelium; an exponential modelwas used for thymus and liver.

For intestinal epithelial cells, a life-span (linear) kinetic model wasemployed based on the assumption that new cells divided, lived for afixed period of time and then died in the order in which they wereformed, representing the progression of intestinal epithelial cells fromcrypt to villus tip (Lipkin, 1987, Physiol. Gastrointest. Tract, pp.255-284, Ed. Johnson, L. R.). A turnover time of 2.8±0.4d was calculated(uncorrected, using plasma glucose enrichments to representintracellular dATP) or 1.8±0.2d (corrected, using plasma glucose with a35% intracellular dilution factor). For thymus and liver a randomreplacement (exponential) model was applied. Thymus had a 10-times morerapid turnover than liver (Table 3).

6.2.4. Clinical Studies of Granulocyte Kinetics in Human Subjects

As part of a study of T lymphocyte kinetics in AIDS, three HIVseropositive men and one HIV seronegative man received an infusion of[6,6-²H₂] glucose (1.25 g/hr for 48 hr). All were clinically stable atthe time of investigation. Absolute granulocyte counts were 1.5, 0.9,and 2.4×10⁹/L, respectively, in the HIV-positive subjects and 2.4×10⁹/Lin the control subject. The infusions were well tolerated. Mean plasmaglucose enrichments of 15.3±2.4 molar percent excess were achieved (rateof appearance of glucose about 2 mglkglmin). Granulocytes were isolatedand dA enrichment measured from DNA. For the first 6 days following theinfusion, very low proportions of labeled cells were seen in thecirculation (FIG. 7), followed by the appearance of labeled cellsstarting on days 6-8. Enrichments at day 8 indicated about 25% new cellspresent (corrected).

6.2.5. Measurement of T Cell Proliferation in HIV Infection

T cell proliferation rates in individuals infected with humanimmunodeficiency virus (HIV) was measured by the methods of theinvention. An intravenous infusion of [6,6-²H₂] glucose was performed inmen with well-maintained CD4⁺ T cell numbers (>500/mm³) or low CD4⁺counts (<200/mm³). The infusion was for 48 hr at 1.25 g [6,6-²H₂]glucose/hr, to achieve 10-15% proportion of labeled glucose molecules inthe blood plasma (10-15% enrichment). Blood (20-30 cc) was collecteddaily during the infusion and for the following 10 days.

Mononuclear cells were isolated using PT® tubes, and CD4⁺ cells werethen isolated using either magnetic bead immunoseparation (Dynabeads®)or fluorescent activated cell sorting to isolate 106 cells. DNA fromisolated cells was recovered using a commercial kit (Quiagen®). DNA washydrolyzed to free deoxyribonucleosides enzymatically with nuclease P₁,phosphodiesterase, and alkaline phosphatase; the hydrolysate wasderivatized with FSTFA to the trimethylsilyl derivatives ofdeoxyribonucleosides, which were injected into a table top GC-MSinstrument. The dA and dG peaks from the GC effluent were monitored byselected ion recording mass spectrometry and m/z 467 and 469 (for dA)and m/z 555 and 557 (for dG) were quantified, through comparison tostandard curves of commercially labeled material analyzed concurrently(e.g., [5,5-²H₂] dA purchased from CIL, Cambridge, Mass.). Standardcurves were abundance matched between standards and sendes to correctfor concentration sensitivity of isotope ratios. Enrichments in dA anddG from CD4⁺ and CD8⁺ T cells were in the rate 0.00 to 1.50 percentlabeled species. By comparison to the plasma glucose isotope enrichment(10-15 percent labeled species) with a 35% dilution correction andapplication of the precursor-product relationship (Hellerstein andNeese, 1992, Am. J. Physiol. 263:E988-1001), the preparation of newlysynthesized DNA strands was quantified. Peak values were at 2-3 daysafter the start of [6,6-²H₂] glucose infusions and reached 15-20% newlysynthesized DNA strands, and thus 15-20% newly proliferating cells (FIG.8). The die away curves of dA or dG labeling between days 4 and 10revealed the destruction rate of the label and therefore recentlydividing population of cells (Hellerstein and Neese, 1992, Am. J.Physiol. 263:E988-1001). Destruction rates of labeled cells weregenerally higher than for the general population of cells implyingselective death of recently divided and activated cells. The effect ofCD4⁺ T cell proliferation and destruction of anti-retroviral therapieswas then determined by repeating the [6,6-²H₂] glucose infusion after8-12 weeks of therapy.

In conclusion, a method for measuring DNA synthesis using stable isotopelabels and mass spectrometry was developed for measuring cellproliferation. This method involves no radioactivity and potential toxicmetabolites, and is thus suitable for use in humans.

6.3. Measurement of T Lymphocyte Kinetics in Humans: Effects of HIV-1Infection and Anti-Retroviral Therapy

The T lymphocyte pool, like all biochemical and cellular systems, existsin a dynamic state: cells die and are replaced by newly divided cells.Depletion of the CD4⁺ T cell pool occurs in HIV-1 infection, but thedynamic basis of this change remains controversial.

A high-turnover kinetic model of CD4 depletion in AIDS has been proposed(Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature373:117-122). The central assertions of this model are that HIV-1 causeshigh rates of CD4⁺ T cell destruction (1-2×10⁹ cells/day) and that thehigh demand on CD4 regenerative systems results, after many years, inexhaustion of lymphopoietic reserves and collapse of the CD4⁺ T cellpool. This model has served as a powerful stimulus for subsequentresearch, but is based on indirect evidence: following initiation ofhighly active anti-retroviral therapy (HAART) in advanced HIV-1 disease,CD4⁺ T cells accumulate in the blood at a rate of 4-8 cells/μL/day.Extrapolating this value to a whole body accumulation rate of 1-2×10⁹cells/day and assuming that the rate of T cell accumulation aftertreatment mirrored the rate of T cell destruction prior to treatment(i.e., that anti-retroviral therapy completely inhibited T celldestruction and had no effect on proliferation), the authors of thismodel concluded that late-stage disease was associated with a very highrate of T cell turnover.

Several investigators have since pointed out, however, that changes incirculating CD4⁺ T cell numbers might represent changes in distributionbetween tissues and blood, due to “viral trapping,” cytokines, stresshormones or other factors, rather than changes in the turnover(proliferation and destruction) of T cells (Dimitrov and Martin, 1995,Nature, 375:194; Mosier, 1995, Nature, 375:193; Sprent and Tough, 1995,Nature, 375:193). If so, inferences about proliferation and destructionwould not be justified from measurement of circulating cell numbersalone. Subsequent studies of the CD4⁺ T cell content of lymphoid tissuesfollowing HAART have confirmed that T cell distribution may indeed bealtered by anti-retroviral therapy and that the initial increase inblood CD4 count might primarily represent redistribution rather thanwhole body T cell accumulation (Zhang et al., 1998, Proc Natl. Acad.Sci. USA 95:1154; Gorochov et al., 1998, Nature Med. 4:215; Bucy et al.,1998, 5th Conference on Retroviral and Opportunistic Infections, Abstr.519:177). Moreover, other workers have concluded, using another indirectmethod for estimating T cell kinetics (i.e., the terminal restrictionfragment (TRF) length of T cell chromosomes) that a high-turnover statedoes not exist in HIV-1 infection (Wolthers et al., 1996, Science274:1543; Palner et al., 1996, J. Exp. Med. 185:1381). The use of TRFshortening rates as an index of replicative history can also becriticized, however, particularly for use in HIV infection (Hellersteinand McCune, 1997, Immunity 7:583).

The absence of quantitative data concerning T cell dynamics in normalhumans has further contributed to uncertainty regarding the T celldynamic consequences of HIV-1 infection. It is not clear, for example,whether a proliferation rate of 1-2×10⁹ cells/day (4-8 cells/μLblood/day) for CD4⁺ T cells (Ho et al., 1995, Nature 373:123-126; Wei etal., 1995, Nature 373:117-122; Perelson et al., 1996, Science 271:1582;Perelson et al., 1997, Nature 387:188; Wain-Hobson, 1995, Nature373:102), even if it were correct, would represent a higher than normalvalue and therefore an unusual proliferative burden on the Tlymphopoietic system.

These uncertainties about T cell dynamics are largely due to limitationsof previous methodology. Until recently, no technique for directly andaccurately measuring T cell kinetics had existed for use in humans. Withthe methods of the present invention, the proliferation and replacementrates of cells in subjects, including humans, can be measured. Themethods of the present invention allow measurement of DNA replicationand cell proliferation by endogenous labeling with stable isotopes (FIG.1), followed by isolation of cellular DNA, enzymatic hydrolysis todeoxyribonucleosides and analysis of isotope enrichment by gaschromatography/mass spectrometry (GC-MS), without involving use ofradioactivity or potentially toxic metabolites. By combining thesetechniques with fluorescence-activated cell sorting (FACS) to purifyselected cell subpopulations (FIG. 9A), the proliferation rate andsurvival time of T cells can be measured in humans.

In this example, we measured the kinetics (proliferation and replacementrates) of circulating T cells in humans with and without HIV-1 infectionusing this stable isotope/FACS/GC-MS method of the present invention. Wefocused on certain fundamental questions regarding T cell dynamics: whatare normal proliferation rates and fractional replacement rate constantsfor circulating CD4⁺ and CD8⁺ T cells in HIV-1-seronegative humans? Arethese values altered in HIV-1 infected patients with measurable plasmaviral load? What are the effects of highly active anti-retroviraltherapy (HAART) regimens after either short-term (e.g., 3 months) orlong-term (e.g., >12 months) therapy? What is the relation between CD4⁺and CD8⁺ T cell kinetics in these settings?

6.3.1. Methods

6.3.1.1. Human Subjects

Kinetic measurements were performed in four groups of subjects (Table4). All subjects were volunteers, recruited by advertisement or word ofmouth:

(I) Normal, HIV-1-seronegative subjects (n=9; 6 men, 3 women). Subjectswere healthy, weight-stable, afebrile and not taking any medications.

(II) HIV-1-infected subjects not receiving protease inhibitor therapyand exhibiting measurable plasma viral load (HIV+ group, n=6; 5 men, 1woman). Subjects had not taken protease inhibitors previously, wereclinically stable, and afebrile. CD4 counts at the time of the study areshown (Table 4).

(III) Subjects studied after 12-weeks of ritonavir/saquinavir therapy incombination with nucleosides (short-term HAART group, n=8 men).Eligibility criteria for enrollment were measurable viral load onnucleoside or non-nucleoside therapies (n=6) or on a protease inhibitorwith nucleosides (n=2); clinical stability; no other medical conditions;and willingness to be followed for 12 weeks after startingritonavir/saquinavir therapy. Nine subjects were enrolled; eightcompleted the 12-week study. Baseline and 12-week T cell counts areshown (Table 4).

(IV) Subjects who had been on a HAART regimen for 12-24 months, withviral load persistently below detection limits (long-term HAART group,n=5 men). These subjects were clinically stable and had documentedsuppression of viral load for 12-24 months. CD4 counts at each patient'snadir and at the time of the study are shown (Table 4).

Written informed consent was obtained from all subjects prior to anyprocedures. The protocol was approved by the UC San Francisco Committeeon Human Research.

TABLE 4 T Cell Kinetics in Individual Subjects CD4 CD8 Group/ CD4 VL CD4CD8 k Abs Prolif Abs Prolif k Abs Prolif Abs Prolif Subject Nadir (×10³)Count ΔCD4 Count (d⁻¹) (cells/μL/d) (cells/d × 10⁻⁹) (d⁻¹ ) (cells/μL/d)(cells/d × 10⁻⁹) I) Normal controls #1. — — 1,792 — 403 0.0120 21.5 5.40.0080 3.2 0.81 #2. — — 875 — 375 0.0084 7.4 1.8 0.0011 0.4 0.10 #3. — —1,509 — 574 0.0046 6.9 1.7 0.0039 2.2 0.56 #4. — — 1,576 — 989 0.014022.1 5.5 0.0110 10.9 2.7 #5. — — 1,317 — 594 0.0046 6.1 1.5 0.0422 25.16.3 #6. — — 891 — 226 0.0035 3.1 0.8 0.0035 0.8 0.2 #7. — — 1,142 — 6640.0059 6.7 1.7 0.0080 5.3 1.3 #8. — — 1,971 — 902 0.0083 16.4 4.1 0.00343.1 0.8 #9. — — 629 — 833 0.0190 12.0 3.0 — — — Mean ± S.D. — —   1300 ±—   618 ±   0.0089 ±   11.4 ±   2.8 ±   0.0100 ±   6.4 ±   1.6 ± 452 257.0052 7.0 1.8 .0130 8.3 2.1 II) HIV −+ #1. — 2.0 183 — 361 0.032 5.9 1.50.023 8.3 2.1 #2. — 21.5 740 — 1173 0.026 19.2 4.8 0.031 36.4 9.1 #3. —107.4 168 — 838 0.040 6.7 1.7 0.048 40.2 10.1 #4. — 43.0 236 — 18590.034 8.0 2.0 — — — #5. — 10.0 636 — 1145 0.022 14.6 3.7 — — — #6. — 440172 — 702 0.031 5.3 1.3 — — — Mean ± S.D. — 102.4 ±   356 ± —   1.031 ±  0.030 ±   9.9 ±   2.5 ±   0.034 ±   28.3 ±   7.1 ± 170.7 261 512 .0065.7 1.4 .013 17.4 4.4 III) Short-Term HAART A) Viral responders #1. 135<500 296 3.1 920 0.078 23.1 5.8 0.084 76.8 19.0 #2. 335 <500 629 4.61,058 0.044 27.6 6.9 0.062 65.6 16.4 #3.  77 <500 363 5.6 987 0.036 11.02.8 0.036 35.2 8.8 #4.  83 <500 135 0.6 776 0.032 4.4 1.1 0.042 32.7 8.2#5. 289 <500 365 2.0 2,277 0.082 29.9 7.5 0.036 81.4 20.0 Mean ± 184 ±<500 ±   358 ±   3.2 ±   1.204 ±   0.053 ±   19.2 ±   4.8 ±   0.052 ±  58.3 ±   14.5 ± S.D. 120 178 2.0 609 0.025 11.0 2.8 .021 23.0 .56 B)Virologic failures #6. 320 47.6 450 0 1,031 0.105 47.1 12.0 0.068 70.217.6 #7.  76 26.0 65 0 563 0.060 4.2 1.1 0.057 32.1 8.0 #8. 164 65.0 1780 1,037 0.036 6.3 1.6 0.026 26.6 6.6 Mean ± 187 ±   46.2 ±   231 ±   0 ±  877 ±   0.067 ±   19.2 ±   4.9 ±   0.050 ±   43.0 ±   10.7 ± S.D. 12419.5 198 0 272 .035 24.2 6.2 .022 23.7 6.0 Total Group, 185 ± —   310 ±  2.0 ±   1,081 ±   0.058 ±   19.2 ±   4.9 ±   0.051 ±   52.6 ±   13.1 ±Mean ± 112 183 2.2 512 .028 15.4 3.9 .020 23.0 5.7 S.D. ,1/11 IV)Long-Term HAART #1.  13 <500 608 — 1,976 0.0091 5.5 1.4 0.0134 26.4 6.6#2. 603 <500 1261 — 880 0.0018 2.3 0.6 0.0027 2.4 0.6 #3.  7 <500 364 —499 0.0103 3.7 0.9 0.0144 7.2 1.8 #4.  87 <500 330 — 898 0.0160 5.3 1.30.0048 4.3 1.1 #5. 381 <500 917 — 943 0.0078 7.2 1.8 — — — Mean 218 ±<500   696 ± —   1039 ±   0.0090 ±   4.8 ±   1.2 ±   0.0088 ±   10.1 ±  2.5 ± 264 394 553 .0051 1.9 0.5 .0059 11.1 2.8 Legend: Abs prolif,absolute proliferation rate; VL, viral load. Plasma HIV-1 viral load wasmeasured by the Chiron bDNA method. CD4+ and CD8+ T cell counts weredetermined by FACS at week 12. CD4 nadir, lowest blood CD4 countdocumented in subject's medical chart. ΔCD4, change in blood CD4 countfrom pretreatment value during first six weeks of short-term HAART(Group III only); —, not measured or not appropriate; “d” represents“day”.

6.3.1.2. Measurement of T Cell Kinetics

Methods of the present invention relating to stable isotope/MS methodsfor measuring cell proliferation and turnover, as described above andherein, were employed for these studies. In brief, such methods involvedthe following four steps:

(1) Administration of [6,6-²H₂] glucose in vivo. 48-hr constantintravenous infusions of labeled glucose were performed to ensure arepresentative time sample of T cell kinetics. Enrichments of plasmaglucose were measured every 12 hr during the infusion. Infusions wereperformed at the General Clinical Research Center of San FranciscoGeneral Hospital. Plasma [²H₂] glucose enrichments decay to <10% ofsteady-state values within 1-2 hr of discontinuing the intravenousinfusion.

(2) Isolation of CD4⁺ and CD8⁺ T cell populations from blood. Blooddraws were generally performed twice (50-70 ml each)—once between days5-7 and once between days 10-14 after the initiation of [²H] glucoseinfusions. Peripheral blood mononuclear cells were initially separatedinto CD4⁺ and CD8⁺ T cell subpopulations using immunoaffinity beads[Dynabeads (Dynal; Oslo, Norway) or MACS separation columns (MiltenyiBiotec, Auburn, Calif.)]. On re-analysis, these cell preparations werefound to be, on average, only 70-90% pure and yielded inconsistentkinetic measurements by GC-MS. Accordingly, it was necessary to isolateCD4⁺ and CD8⁺ T cell subpopulations to >98% purity using multiparameterflow cytometry (FIG. 9A).

(3) Preparation of dA from T cell DNA. Purine deoxyribonucleosides (dAand dG) were isolated from T cell DNA for subsequent GC-MS analysis(FIG. 9B). In general, ≧5 μg of DNA, representing roughly 10 cells, wererequired to recover sufficient dA for GC-MS measurements.

(4) Mass spectrometric measurements of ²H-enrichments in dA. GC-MSanalysis of isotope enrichments of dA was performed by comparison tostandard curves of [²H₂]dA (FIG. 9C).

6.3.1.3. Calculations of T Cell Kinetic Parameters

Kinetic calculations are based on the mathematics of theprecursor-product relationship, or Newton's cooling equation,representing exchange or replacement of unlabeled by labeled material asdescribed herein (Macallan et al., 1998 Proc. Natl. Acad. Sci. USA95:708-713; Zilversmit et al., 1974, J. Gen. Physiol. 26:325;Hellerstein and Neese, 1992, Am. J. Physiol. 263:E988-E1001; Waterlow etal., PROTEIN TURNOVER IN MAMMALIAN TISSUES AND IN THE WHOLE BODY 216-219(North-Holland Publishing Co., Amsterdam, 1978)). The fractionalreplacement rate (k, d⁻¹), or the rate constant of input and removalunder the steady-state or pseudo-steady-state conditions present, iscalculated from the ratio of label incorporation into product (T celldA) compared to precursor (blood glucose, corrected for intracellulardilution in the deoxyribonucleoside pool):

Equation (1):

dB/dt=k(A−B),

where A is the isotope enrichment of the precursor and B is the isotopicenrichment of the product.

When A is constant, integration yields:

Equation (2):${{{Equation}\quad (2)}:{B/A}} = {\frac{\left\lbrack {}^{2}H_{2} \right\rbrack {dA}\quad {enrichment}}{\left\lbrack {}^{2}H_{2} \right\rbrack \quad {Glucose}\quad {enrichment} \times 0.65} = {1 - e^{- {kt}}}}$

Rearranging,

Equation (3):

k= ⁻ In(1−[B/A])/t

where t is 2 days (the isotope labeling period) and enrichmentrepresents the fraction of [²H₂] labeled molecules present. Absolute Tcell proliferation rates are then calculated as (k× pool size), wherepool size equals measured blood count (T cells/μL). Extrapolation towhole-body T cell absolute proliferation rates can then be performed asdescribed elsewhere (Ho et al., 1995, Nature 373:123-126; Wei et al.,1995, Nature 373:117-122) by multiplying×10⁶ μL/L)×(5 L bloodvolume)×(50).

These calculations are based on the following: circulating T cell countsduring the labeling period are stable, so that entry of newly dividedcells into the circulation must be balanced by exit of other cells(steady-state assumption); proliferating T cells and non-proliferating Tcells traffic similarly between tissues and blood, so that the fractionof newly divided cells in blood represents that in tissues; and themetabolic contribution from extracellular glucose to the deoxyribosemoiety of DATP in T cells (FIG. 1) is uniform in all lymphoproliferativetissues and is at the value calculated previously, in vitro, herein(Table 2; see also Macallan et al., 1998 Proc. Natl. Acad. Sci. USA95:708-713). Interpretation of replacement rates of circulating T cellsas representative of T cell destruction assumes that tissue T cells poolsize is constant during the labeling period, so that production of newlydivided cells must be balanced by destruction of other cells.Extrapolation of absolute proliferation rates of circulating T cells tothe whole body assumes that the distribution ratio between tissues andblood is constant and is at the value (50:1) estimated elsewhere (Ho etal., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122)1).

6.3.1.4. Statistical Analyses

Groups were compared by one-way ANOVA with Dunn/Bonferroni follow-up ata procedure-wise error-rate of 5%. CD4⁺ were compared to CD8⁺ T cellswithin groups by paired T-test.

6.3.2. Results

Measurement of dynamics of circulating lymphocytes has a somewhatatypical feature in that the compartment sampled (blood) is not thecompartment where cell proliferation is believed to occur.Interpretation of label incorporation in circulating T cells must takethis compartmentalization into account. The results in FIG. 10 provideinsight into the time course of label incorporation as measured incirculating CD4⁺ T cells. In this group of 8 subjects on short-termHAART (Group III), most (6) showed equivalent levels of labelincorporation at day 5 and day 14; three subjects with samples also atdays 6 or 7 demonstrated stability of label incorporation during thisinterval. These observations are consistent with a model of T cellproliferation in a central, unsampled compartment followed by rapidexchange (mixing) into the circulating pool over the subsequent 5-14days. Because the blood CD4 pool recirculates several times per day(Gowans et al., 1998, Blood 91:1653), apparent stability of enrichmentsin circulating T cells (FIG. 10) indicates that the exchanging pools arewell mixed. The lack of a fall-off in CD4⁺ T cell enrichment over thetwo weeks following cessation of label likely reflects residual slowentry of labeled cells from tissues, counterbalancing destruction oflabeled cells in the circulation. If blood and tissues are less than100% mixed or if some labeled cells are destroyed before appearing inthe bloodstream, the measured replacement rate in blood will thereforeunderestimate true rates of tissue T cell proliferation, although theywill still accurately reflect replacement and proliferation of cellspresent in the bloodstream. The turnover rates measured should thus beviewed as minimum values, when extrapolating to the tissues.

In two subjects studied in Group III, measured incorporation increasedbetween the first and last time points, although label administrationhad long since ceased. In these instances, proliferating cells (labeledduring the first two days) must have exchanged into the peripheralcirculation at a slower rate than that observed in the six othersubjects. For the purposes of the data presentation below, measuredincorporation values from such individuals were taken to be the datewith the highest value. As above, these results thus represent minimumestimated values of tissue rates of T cell proliferation. Similar timecourses of label incorporation into circulating CD4⁺ T cells wereobserved in subjects from Groups I, II, and IV (Table 4).

6.3.2.1. CD4⁺ and CD8⁺ T Cell Dynamics

The fractional replacement rate (k) for CD4⁺ T cells in normal, HIV-1seronegative subjects was 0.0089±0.0052 d⁻¹ (Table 4, Group I). Thevalue of k for CD8⁺ T cells was 0.0100±0.0130 d⁻¹. Absoluteproliferation rates of circulating T cells were 11.4±7.0 and 6.4±8.3cells/μL/day for CD4⁺ and CD8⁺ T cells, respectively. If standardestimates of tissue:blood T cell distribution (50:1) are applied (Ho etal., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122;Perelson et al., 1996, Science 271:1582: Perelson et al., 1997, Nature387:188; Wain-Hobson, 1995, Nature 373:102), these rates in blood can beextrapolated to 2.8±1.8×10⁹ and 1.6±2.1×10⁹ cells/day, respectively, inthe whole body. In these HIV-1-seronegative subjects, the proliferationrate for CD4⁺ T cells is thus higher than that found for CD8⁺ T cells bya factor of almost two. Notably, the CD4⁺ turnover rate in theseHIV-1-seronegative subjects is also higher than that previouslyestimated, using indirect means, to exist during late-stage HIV-1disease (Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature373:117-122).

Kinetic results in HIV-1-seropositive men (n=6) with measurable plasmaviral loads (mean 10^(5.0±5.2); CD4 count 360±267) are shown (Table 4,Group II). The value of k for CD4⁺ T cells was about 3-fold higher inHIV-1-infected subjects (0.031±0.006 d⁻¹) compared to normal controls(FIGS. 11A and 11B). The absolute proliferation rate of circulating CD4⁺T cells, 9.9±5.7 cells/μL/day, representing the entry of newly dividedcells into the circulating pool (or a whole body rate of 2.5±1.4×10⁹cells/day) was not elevated above normal in uncontrolled HIV-1 infection(FIGS. 11A and 11B). In contrast, the absolute rate of circulating CD8⁺T cell proliferation was elevated in HIV-1 infection (28.3±17.4cells/μL/day or 7.1±4.4×10⁹ cells/day in the whole body, FIGS. 11A and11B) compared to normal controls, and was greater, rather than less,than that found for CD4⁺ T cells.

Eight subjects were studied after being placed on a combined proteaseinhibitor—containing HAART regimen (ritonavir/saquinavir, added toprevious nucleoside or non-nucleoside agents) for 12 weeks, at whichtime blood CD4 counts were relatively stable (Table 4; Ho et al., 1995,Nature 373:123-126; Wei et al., 1995, Nature 373:117-122; Perelson etal., 1996, Science 271:1582; Perelson et al., 1997, Nature 387:188;Wain-Hobson, 1995, Nature 373:102; Zhang et al., 1998, Proc Natl. Acad.Sci. USA 95:1154). The measured T cell replacement rates in thebloodstream at this time were compared to the average accumulation rateof CD4⁺ T cells in blood during the first 6 week non-steady-state phase(i.e., inferred from the difference between baseline and week 6 bloodCD4 counts; see FIG. 12 legend). The average accumulation rate duringthe initial non-steady state period underestimated true replacementrates at steady-state and did not correlate well with measured values,even qualitatively (FIG. 12). Some subjects, for example, did notdemonstrate any increase in blood CD4 counts on HAART, so that turnovercould not be estimated by the accumulation method. Nevertheless, thesesubjects exhibited active turnover of CD4⁺ T cells at 12 weeks oftherapy (FIG. 12).

Compared to both HIV-1-seronegative controls and HIV-1-infected subjectsnot on protease-inhibitor-containing regimens, a number of significantdifferences in T cell kinetics were documented in patients after ashort-term HAART regimen (FIGS. 11A and 11B). Whether or not the viralload decreased, the values for k and the absolute proliferation rates ofCD4⁺ and CD8⁺ T cells were generally higher. T cell kinetics in GroupIII were not noticeably different for virologic responders (viralload<500 copies/ml) and virologic failures (viral load>500 copies/ml,Table 4, FIGS. 11A and 11B). Subjects on long-term HAART (Group IV, withsustained suppression of viral load for 12-24 months) exhibited stilldifferent kinetics (FIGS. 11A and 11B): In this group of 5 individuals,the values for k and the absolute proliferation rates for CD4⁺ and CD8⁺T cells were essentially back-to-normal values observed inHIV-1-seronegative subjects.

Strong correlations between CD4⁺ and CD8⁺ T cell absolute proliferationrates (FIG. 13A, r²=0.69, p<0.001) and k (FIG. 13B, r²=0.66, p<0.001)were observed. A strong correlation between absolute proliferation rateand blood count of CD4⁺ T cells in both HIV-1 seropositive (FIG. 13C,r²=0.96, p<0.0001) and short-term HAART groups (FIG. 13D, r²=0.55,p<0.01) was also observed, whereas no correlation between k and CD4count (FIG. 13E) was present. There was no correlation between plasmaviral load and either absolute proliferation rate (FIG. 13F) or k (FIG.13G) of circulating CD4⁺ T cells in the HIV-1-infected groups, i.e.,high plasma viral load was not associated with high rates of CD4⁺ T cellturnover, either fractional or absolute, in the bloodstream.

6.3.3. Analysis

T lymphocyte kinetics can be directly measured in human subjects usingthe stable isotope/FACS/GC-MS methods of the present invention describedherein and above. A number of questions concerning theimmunopathogenesis of HIV-1 can thereby be addressed.

Question (1): What are the kinetics of turnover (rate constant ofreplacement and absolute proliferation rate) of circulating T cells innormal humans?

Absent information about circulating T cell turnover rates inHIV-1-uninfected normal controls, it is not possible to conclude thatsuch rates are altered during the course of HIV-1 disease. Indirectmethods have previously been used to assess the normal turnover rates,including measurement of the persistence of unstable chromosome damagein T cells following radiotherapy (McLean and Michie, 1995, Proc. Natl.Acad. Sci. USA 92:3707; Michie et al., 1992, Nature 360:264). Estimatesobtained by this approach (k=0.01 d⁻¹ for memory T cells and 0.001 d⁻¹for naive T cells) are close to, though slightly lower than, themeasured values described herein of ca. 0.009 d⁻¹ for the mixed T cellpool. This slightly lower daily turnover estimate (4-8 cells/μL/day vs.11 cells/μL/day measured by our approach) may reflect prolonged T cellsurvival in the lymphopenic “normal” subjects following radiationtherapy (McLean and Michie, 1995, Proc. Natl. Acad. Sci. USA 92:3707;Michie et al., 1992, Nature 360:264).

In the context of HIV-1 infection, neither the results described hereinnor previous results (McLean and Michie, 1995, Proc. Natl. Acad. Sci.USA 92:3707; Michie et al., 1992, Nature 360:264) support the view thata CD4⁺ T cell regeneration rate of 4-8 cells/μL/day (1-2×10⁹ cells/day)in AIDS is unusually high. Models assuming that proliferation rates inthe circulating CD4⁺ T cell pool in this range place a chronic strain onlymphopoietic reserves (Ho et al., 1995, Nature 373:123-126; Wei et al.,1995, Nature 373:117-122; Perelson et al., 1996, Science 271:1582;Perelson et al., 1997, Nature 387:188; Wain-Hobson, 1995, Nature373:102) are therefore not consistent with available reference data fornormal humans.

Question (2): Is CD4⁺ T cell depletion in advanced HIV-1 disease due toaccelerated destruction (high-turnover model), regenerative failure(low-turnover model), or both?

It is important to define explicitly how the term “turnover” is beingused, because this term is often used to signify two different aspectsof the process of cell replacement. “Turnover” can represent either: (i)the absolute rate at which cells are formed and die (absoluteproliferation rate, cells/day); or (ii) the fraction of the pool ofcells that is replaced per day (k, d⁻¹). In biochemical systems, poolsize is typically determined by the absolute production rate (generallyzero-order with respect to the end-product) and the rate constant forremoval (which is generally first order with respect to the end-product)(Waterlow et al., PROTEIN TURNOVER IN MAMMALIAN TISSUES AND IN THE WHOLEBODY 198-211 (North-Holland Publishing Co., Amsterdam, 1978); Schimke,in MAMMALIAN PROTEIN METABOLISM 178-228 (H. N. Munro ed., Acad. Press,New York, 1970)). The relevant question in HIV-1 infection is whetheraccelerated destruction and/or impaired production of T cells drivesCD4⁺ T cell depletion. The two models have opposite kinetic predictions:an accelerated destruction model (Ho et al., 1995, Nature 373:123-126;Wei et al., 1995, Nature 373:117-122; Perelson et al., 1996, Science271:1582; Perelson et al., 1997, Nature 387:188; Wain-Hobson, 1995,Nature 373:102) predicts high turnover of CD4⁺ T cells in untreatedHIV-1 disease, an inverse correlation between turnover and the CD4count, and a reduction in the turnover rate after HAART (see FIG. 14,left). In contrast, a regenerative failure model (Wolthers et al., 1996,Science 274:1543; Palmer et al., 1996, J. Exp. Med. 185:1381;Hellerstein and McCune, 1997, Immunity 7:583) predicts a low CD4⁺ T cellturnover rate in untreated HIV-1 disease, a direct correlation betweenturnover and the CD4 count, and an increase in the turnover rate afterinitiation of HAART (see FIG. 14, right).

Although k for CD4⁺ T cells was 3 times higher in HIV-1-positive than inHIV-1-negative subjects, observations suggest that the T cell productionrate or regenerative capacity plays the quantitatively more importantrole in determining circulating CD4⁺ T cell counts, at least in theadvanced HIV-1 populations that were studied herein:

(i) Short-term HAART was associated with higher, not lower, values ofboth fractional and absolute replacement rates for circulating CD4⁺ Tcells (FIGS. 11A and 11B). These data demonstrate that the steady-stateincrease in CD4 counts observed after short-term HAART is not due tolonger survival of circulating CD4⁺ T cells (in fact, survival time orhalf-life is shorter), but reflects instead an increased rate of entryof newly produced T cells into the circulating pool.

(ii) The absolute proliferation rates of circulating CD4⁺ T cells werenot higher in HIV-1-infected subjects with high viral load (FIG. 13F),and were not higher in untreated HIV-1-seropositive subjects compared tonormal, seronegative controls (Table 4, FIG. 11B). These observations donot support the model of a viral-driven lymphopoietic burden—at least inadvanced HIV-1 disease.

(iii) The higher the absolute turnover rate of blood CD4⁺ T cells, thehigher was the CD4 count, but there was no correlation with k (FIGS.13C-13E). This result is opposite to the kinetics predicted by theaccelerated destruction model (FIG. 14), according to whichHIV-1-seropositive individuals with the most rapid CD4⁺ T cell turnovershould have the lowest CD4 counts (compare FIGS. 13C and 13D to FIG.14).

(iv) Short-term HAART increased proliferation of CD8⁺ T cells to thesame extent as CD4⁺ T cells (FIGS. 11A and 11B) and HIV-1 infectedsubjects with low CD4 proliferation rates also had low CD8⁺proliferation rates (FIG. 13A). Thus, CD8⁺ T cells shared an apparentregenerative limitation with CD4⁺ T cells in advanced HIV-1 disease.

These observations indicate that regeneration of both CD4⁺ and CD8⁺ Tcells is limited in HIV-1 disease and that such limitation is relievedby anti-retroviral therapy. Recent observations (Zhang et al., 1998,Proc Natl. Acad. Sci. USA 95:1154) on peripheral lymphoid tissues areconsistent with the view that T cell regenerative systems improve afterHAART. Partial restoration of follicular dendritic networks occursrapidly in lymphoid tissue, with appearance of nascent lymphoidfollicles and cellular infiltrates (Zhang et al., 1998, Proc Natl. Acad.Sci. USA 95:1154). The lower the initial CD4 count, the more dramaticwere the changes in lymphoid architecture. The kinetic data presentedherein support the notion that the microenvironment for T cellproliferation in peripheral lymphoid tissues and perhaps thymus can beimproved by anti-retroviral therapy.

Question (3): Is there evidence for “blind homeostasis” for T cells inHIV-1 infection?

A “blind homeostasis” model has been proposed (Roederer, 1995, NatureMed. 1(7):621; Margolick et al., 1995, Nature Med. 1(7):674-680) toexplain reciprocal changes in circulating CD4⁺ and CD8⁺ T cell countsduring the course of HIV-1 disease. This model postulates a T cellcounter that does not distinguish between CD4⁺ and CD8⁺ T cells: if CD4⁺T cells are destroyed, then CD8⁺ T cells are produced in greaterquantities to fill the available T cell space. In ecologic terminology,CD4⁺ and CD8⁺ T cell regeneration are proposed to be in competition forshared and limiting resources (e.g., antigen-presenting cells,co-stimulatory molecules, etc.).

The data presented herein are partly consistent with this model. Infavor of the model is the observation that the ratio of CD8:CD4 absoluteproliferation rates reverses after infection with HIV-1 (Table 4, FIG.11B). In normal subjects, the absolute proliferation rate or circulatingCD4⁺ T cells was twice as high as the absolute proliferation rate ofcirculating CD8⁺ T cells, while in HIV-1-infected groups, the CD8 ratewas at least twice as high as the CD4 rate, paralleling changes in CD8⁺T cell pool size in blood (Table 4).

On the other hand, treatment with HAART was associated with coordinateincreases in both CD4⁺ and CD8⁺ T cell absolute proliferation rates(FIGS. 11A and 11B). Moreover, within HIV+ groups, there was a strongdirect correlation between CD4⁺ and CD8⁺ proliferation rates (FIG. 13A).These observations indicate that, at least in the setting of late-stageHIV-1 disease, the production of both CD4⁺ and CD8⁺ T cells is limitedby the absence of shared factors.

These results indicate that an element of competition for resources ispresent in advanced HIV-1 disease, but that common regenerative defectsare superimposed. Longitudinal studies of CD4 and CD8 kinetics over thenatural history of HIV-1 infection are required to resolve thisquestion.

Question (4): Is most CD4 turnover due to direct HIV-1-mediated killing?

Although it is clear that HIV-1 can directly infect and destroy CD4⁺(and possibly CD8⁺ T cells, Flamand et al., 1998, Proc. Natl. Acad. Sci.USA 95:3111) in vivo, kinetic analysis shows that most death ofcirculating CD4⁺ T cell occurs in a manner which is not correlateddirectly with circulating HIV-1:

i) In the five “viral responders” on short-term HAART (Table 4, GroupIIIA), the k for CD4⁺ T cells was higher, not lower, than that found inHIV-1-infected subjects with measurable viral load (Group II)—i.e., ahigher fraction of CD4⁺ T cells were dying in the face of a lowercirculating viral load (FIG. 11B). This result may reflect disinhibitionof T cell regeneration, leading over the short-term to increased levelsof CD4⁺ T cell proliferation and, thus, an increased level ofactivation-induced cell death (AICD) (Murali-Krishna et al., 1998,Immunity 8:177). Lower viral loads may initially increase total levelsof CD4⁺ T cell death, because more CD4⁺ T cells are being produced.

ii) A similar observation holds for the turnover of CD8⁺ T cells: Thesecells have a higher k, or a shorter survival, in “viral responders”(Group III) than in HIV-1-infected subjects with active viralreplication (Group II). The half-lives (1/k) for CD8⁺ T cells wereidentical to those for CD4⁺ T cells across the different HIV-1-infectedgroups (FIG. 11A) and were strongly correlated within groups (FIG. 13B).If CD8⁺ T cells are not infected and destroyed in vivo, it is difficultto attribute their elevated values for k and absolute proliferation rateto a direct effect of HIV-1.

iii) Finally, there was found to be no correlation between circulatingviral load and CD4⁺ T cell proliferation rates or k (FIGS. 13F and 13G).Plasma viral load has previously been interpreted as an index of therate of CD4⁺ T cell destruction in the body (Perelson et al., 1996,Science 271:1582; Perelson et al., 1997, Nature 387:188; Wain-Hobson,1995, Nature 373:102; Mellors et al., 1996, Science 272:1167; Mellors etal., 1995, Ann. Int. Med. 122:573). Although we cannot exclude thepossibility that T cell destruction was occurring in lymphoid tissues,our data do not support the notion that plasma viral loads arereflective of destruction rates of circulating CD4 ⁺ T cells.

These kinetic findings do not argue against the occurrence ofvirally-mediated CD4⁺ (or CD8⁺) T cell killing. Rather, they demonstratethat most dying CD4⁺ (and CD8⁺) T cells in the circulating pool may beuninfected (Oyaizu and Pahwa, 1995, J. Clin. Immunol. 15:217; Casellaand Finkel, 1997, Curr. Op. Hematol. 4:24; Anderson et al., 1998, J.AIDS Hum. Retrovirol. 17:245.

Question (5): What drives T cell proliferation in advanced HIV-1disease?

Comparison of T cell proliferation rates in the three HIV-1-infectedgroups (FIG. 11B) shows that T cell regeneration is largelyantigen-driven in this setting. The extremely high proliferation of bothCD4⁺ and CD8⁺ T cells after short-term HAART (compared to HIV-1 infectedsubjects not on HAART as well as to HIV-1 seronegative controls)contrasts with the strikingly lower turnover—both fractional andabsolute—after 12-24 months of HAART. The latter circumstance mayreflect normalization of the antigenic burden, whereas the formersituation is replete with antigenic stimuli, including HIV-1 itself aswell as pathogenic organisms. This interpretation predicts that theearly increase in T cell proliferation should reflect mostly memory Tcells—i.e., antigen-driven cell proliferation—and should not beassociated with a more diverse T cell receptor (TCR) repertoire (Connorset al., 1997, Nature Med. 3:533). In contrast, proliferation afterlonger-term viral suppression, if it is not antigen driven andparticularly if it involves intrathymic maturation (McCune, 1997, Sem.Immunol. 9:397), may include generation of naive T cells with attendantbroadening of TCR repertoire diversity.

One of the puzzling questions about immune cell dynamics in AIDS hasbeen why CD4 counts increase so quickly after HAART, but recover muchmore slowly after bone marrow transplantation or radiationtherapy-induced lymphopenias (Mackall et al., 1994, Blood 84:2221;Mackall et al., 1997, Immunol. Today 18:245; Mackall et al., 1997, Blood89:3700). The implication that HIV-1 infection somehow stimulates Tlymphopoiesis has been difficult to rationalize biologically. Anantigen-driven model—in combination with a less damaged or morereversibly damaged microenvironment in AIDS—might explain differencesbetween these settings. The replacement rates of roughly 5 to 47 CD4⁺ Tcells/μL/day and 27 to 81 CD8⁺ T cells/μL/day in patients after 12 weeksof combined protease inhibitor treatment (Table 4) are more thansufficient to account for relatively rapid changes in the circulating Tcell pool size in this setting—even if fractional destruction rates arealso elevated. The strikingly lower replacement rates in patients after12-24 months of HAART (2 to 7 CD4 cells/μL/day and 4 to 26 CD8cells/μL/day) may more closely resemble kinetics in the post-bone marrowtransplantation or radiotherapy settings. Measuring lymphocyte dynamicsin these non-HIV-related lymphopenic states is of interest.

Question (6): Does long-term anti-retroviral therapy have differenteffects on T cell dynamics than short-term therapy?

Recent studies have suggested that the phenotypes of T cell populationsare affected differently after 12-18 months of proteaseinhibitor-containing regimens than during the initial 3-6 months oftreatment (Autran et al., 1997, Science 277:112; Connick et al., 1998,5th Conference on Retroviral and Opportunistic Infections, Abstr.LB14:225). Of particular interest, late increases in naive phenotype(CD45RA+CD62L+) T cells have been reported. Our results (Table 4, FIGS.11A and 11B) demonstrate very different T cell kinetics after long-termcompared to short-term therapy. After 12-24 months, fractional andabsolute turnover rates of CD4⁺ T cells were reduced to those of normalcontrols, compared to the much higher values present after three monthsof HAART. In conjunction with reports that kinetics during the initial4-6 weeks after HAART may primarily reflect redistribution betweentissue and blood (Zhang et al., 1998, Proc Natl. Acad. Sci. USA 95:1154;Gorochov et al., 1998, Nature Med. 4:215; Bucy et al., 1998, 5thConference on Retroviral and Opportunistic Infections, Abstr. 519:177),these data show that the post-HAART period can be divisible into atleast three distinct kinetic phases: an initial non-steady state (orredistribution) phase during the first 0-2 months, a period ofaccelerated proliferation and destruction during the 2-6 month period,and a low turnover phase thereafter which is possibly characterized bynaive T cell regeneration and immune reconstitution. This model of Tcell dynamics is testable prospectively, using the methods of thepresent invention described herein.

Question (7): What are the implications for other techniques forestimating T cell kinetics?

The results presented herein—comparing direct measurements of T cellturnover to the accumulation rate of circulating T cells in the initialnon-steady-state (FIG. 12)—indicate that the latter approach is notinformative regarding true turnover even of the circulating T cell pool.Besides under-estimating steady-state turnover, there was no reliablecorrelation with true replacement rates, and effects of redistributionon the early accumulation rate can not be excluded. In addition, theaccumulation method can not be applied if T cell counts are stable, andsome of the more interesting subjects in Group III were in this category(Table 4). The assumption that CD4⁺ T cell destruction is reduced tozero following acute suppression of viral replication (Ho et al., 1995,Nature 373:123-126; Wei et al., 1995, Nature 373:117-122; Perelson etal., 1996, Science 271:1582; Perelson et al., 1997, Nature 387:188;Wain-Hobson, 1995, Nature 373:102) is not consistent with the datapresented herein, since normal HIV-1-negative subjects had a CD4⁺ T cellturnover of about 11.4 cells/μL/day (or 2.8×10⁹ cells/d in the wholebody). Thus, post-HAART CD4 accumulation rates in the bloodstream do notrepresent either pre-HAART or post-HAART proliferation or destructionrates of blood CD4⁺ T cells.

The rate of telomeric TRF shortening has also been used as an index of Tcell replicative history in HIV-1 infection (Wolthers et al., 1996,Science 274:1543; Palmer et al., 1996, J. Exp. Med. 185:1381). Thismethod is especially problematic in HIV-1 infection because of apotential selection bias against proliferating cell populations (seealso Hellerstein and McCune, 1997, Immunity 7:583). The problem is thatif HIV-1 enters and kills activated cells preferentially, the TRFlengths of surviving cells may not reflect the replicative history ofthe general population. Neither Wolthers et al. nor Palmer et al. founda higher rate of TRF shortening for CD4⁺ T cells in HIV-1-infectedpatients compared to HIV-1-seronegative controls (see Wolthers et al.,1996, Science 274:1543; Palmer et al., 1996, J. Exp. Med. 185:1381), butWolthers et al. have since noted that an increase in CD4⁺ T cellfractional turnover up to 3-fold above normal values would still becompatible with their TRF data, if HIV-1 has a 30% efficiency forinfection and destruction of proliferating CD4⁺ T cells (see Wolthers etal., 1998, Immunol. Today L9:44). According to this formulation, ourfinding of a ca. 3-fold higher k in HIV+ subjects is thereforeconsistent with the TRF data. The report (Wolthers et al., 1996, Science274:1543; Palmer et al., 1996, J. Exp. Med. 185:1381)) of higher CD8⁺ Tcell turnover in HIV-1-infected subjects than in HIV-1-seronegativecontrols is also consistent with the results presented herein, ifproliferating CD8⁺ T cells are not preferentially infected and destroyedby HIV-1.

Question (8): Do these findings have any relevance to clinicalmanagement of HIV-1-infected patients?

An important feature of these kinetic results was the markedheterogeneity among individuals in each group, especially in terms ofthe dynamic response to HAART (Table 4). Many factors likely influence Tcell kinetics, including the stage of disease, control of viralreplication, age of the patient, antigenic burden, thymic function, andcapacity of lymphoid tissue to support antigen-dependent T cellregeneration. Heterogeneity within a disease population raises thepossibility of identifying pathogenically distinct clinical subgroups.Certain individuals studied herein suggest that clinical subgroups mayexist and be identifiable. Two examples follow from the short-term HAARTgroup (Group III, Table 4):

Patient #4 (43 year old man with AIDS). Although the plasma viral loadfell from 157,000 to <500 on HAART, there was only a small increase inCD4 count (from 83 to 135 CD4/μL). The absolute proliferation rate inthis subject was only 4.4 cells/μL/d (1.1×10⁹ cells/d) compared to amean of 19.2 cells/μL/d for Group III as a whole. Patients of this typemay reflect more advanced damage to T lymphopoietic systems and may becandidates for adjuvant immunostimulatory therapies (e.g.,interleukin-2, transplantation of thymic tissue or progenitor cells).

Patient #6 (48 year old man with AIDS). This patient had previouslyfailed another protease inhibitor (indinavir, viral load=28,000) andthen had a transient response on ritonavir/saquinavir before failing(viral load=47,000). Nevertheless, he maintained a CD4 count around 450cells/μL and had an absolute proliferation rate of 47.1 cells/IL/d(12.5×10⁹ cells/d). Thus, despite the half-life of his blood CD4 cellsbeing only 6.5 days (k=0.105 d⁻¹), the circulating CD4 pool was beingmaintained by a very high T cell regenerative rate. These findingsreveal a capacity of the T cell regenerative systems that is notapparent from measurement of the plasma viral load and which may evenpoint to a salutary effect of anti-retroviral therapy in the face of“virologic failure” (e.g., perhaps secondary to local effects on Tlymphocyte regeneration, altered pathogenicity of the virus, etc.).

Kinetics also provides clinically useful information regarding the timeto initiate anti-retroviral therapy. If alterations in T cell dynamics(“stress”) precede alterations in T cell pool size (“strain”),measurements of the former can facilitate prevention of the latter.

In summary, the present invention provides methods for measuring T cellkinetics directly in humans. With such methods, fundamental questionsabout the immunopathogenesis of HIV-1 can be addressed. The resultspresented herein focus on T cell regenerating systems in thepathogenesis of HIV-1 disease and in the response to anti-retroviraltherapy. The results also show that mechanisms other than directHIV-1-mediated CD4⁺ T cell killing are the cause of most T cell turnoverin advanced HIV-1 disease and that T cell turnover is antigen-driven inthe post-HAART setting. As will be apparent to those of ordinary skillin the art based on the detailed disclosure provided herein, the methodsof the present invention are broadly applicable to other aspects ofHIV-1 immunopathogenesis and therapy in vivo.

From the foregoing, it should be apparent that many of the describedmethods have several general features which can be expressed conciselyas follows: In vitro and in vivo use of a stable isotope label to labelthe DNA of a cell. In vitro and in vivo use of a stable isotope label tolabel the DNA of a cell via the de novo nucleotide synthesis pathway. Invitro and in vivo use of a stable isotope label to label the DNA of acell via the de novo nucleotide synthesis pathway to measure cellularproliferation and/or cellular destruction rates. Use of a stable isotopelabel and a non-stable radioactive isotope label to label the DNA of acell, including labeling such cell via the de novo nucleotide synthesispathway. Use of a stable isotope label and a radioactive isotope labelto label the DNA of a cell via the de novo nucleotide synthesis pathwayto measure cellular proliferation and/or cellular destruction rates. Useof a stable isotope label incorporated into DNA via the de novonucleotide synthesis pathway to screen an agent for capacity to induceor inhibit cellular proliferation. Use of a stable isotope labelincorporated into DNA via the de novo nucleotide synthesis pathway toascertain the susceptibility of a subject to a disease which inducescellular proliferation or destruction or a change in a rate of cellularproliferation or cellular destruction.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention and any sequences which are functionally equivalent are withinthe scope of the invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

All publications cited herein are incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A double labeling method for measuring cellularproliferation in a proliferating population of cells, said methodcomprising: (a) contacting the proliferating population of cells with adetectable amount of a first label, wherein the first label comprises astable isotope label which is incorporated into DNA via the de novanucleotide synthesis pathway; (b) detecting the first label incorporatedinto the DNA to measure cellular proliferation in the proliferatingpopulation of cells; (c) contacting the proliferating population ofcells with a detectable amount of a second label, wherein the secondlabel comprises a radioactive isotope label which is incorporated intoDNA via the de nova nucleotide synthesis pathway; and (d) detecting thesecond label incorporated into the DNA to measure cellular proliferationin the proliferating population of cells.
 2. The method of claim 1,wherein steps (a) and (b) are performed before steps (c) and (d).
 3. Themethod of claim 1, wherein steps (c) and (d) are performed before steps(a) and (b).
 4. The method of claim 1, herein steps (a) and (c) areperformed simultaneously and steps (b) and (d) are performedsimultaneously.
 5. The method of claim 1, wherein the stable isotopelabel and the radioactive isotope label are each attached to a precursorof deoxyribose in the de nova nucleotide synthesis pathway, each saidprecursor being incorporated into deoxyribose.
 6. The method of claim 5,wherein the stable isotope and the radioactive isotope label eachcomprise a labeled glucose.
 7. The method of claim 1, comprisinghydrolyzing the DNA incorporating the stable isotope label todeoxyribonucleosides prior to detecting the label in the DNA.
 8. Themethod of claim 1, comprising hydrolyzing the DNA incorporating theradioactive isotope label to deoxyribonucleosides prior to detecting thelabel in the DNA.
 9. The method of claim 1, comprising measuring thestable isotope label in intact DNA polymers.
 10. The method of claim 1,comprising measuring the radioactive isotope label in intact DNApolymers.
 11. The method of claim 1, wherein the radioactive isotopelabel is detected by accelerator mass spectrometry, liquid scintillationcounting, or gamma counting.
 12. The method of claim 1, wherein thestable isotope label is detected by mass spectrometry.
 13. The method ofclaim 1, wherein the proliferating population of cells comprises cancercells.
 14. The method of claim 1, wherein the proliferating populationof cells comprises lymphocytes.
 15. The method of claim 14, wherein thelymphocytes comprise CD4⁺cells.
 16. The method of claim 14, wherein thelymphocytes comprise CD8⁺cells.